MRNA therapy for phenylketonuria

ABSTRACT

The present invention provides, among other things, methods of treating phenylketonuria (PKU), including administering to a subject in need of treatment a composition comprising an mRNA encoding phenylalanine hydroxylase (PAH) at an effective dose and an administration interval such that at least one symptom or feature of PKU is reduced in intensity, severity, or frequency or has delayed in onset. In some embodiments, the mRNA is encapsulated in a liposome comprising one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 61/894,303, filed Oct. 22, 2013, the disclosure of which is herebyincorporated by reference.

SEQUENCE LISTING

The present specification makes reference to a Sequence Listing(submitted electronically as a .txt file named “2006685-0688_SL.txt” onOct. 22, 2014). The .txt file was generated on Oct. 20, 2014 and is18,455 bytes in size. The entire contents of the Sequence Listing areherein incorporated by reference.

BACKGROUND

Phenylketonuria (PKU) is an autosomal recessive metabolic geneticdisorder characterized by a mutation in the gene for the hepatic enzymephenylalanine hydroxylase (PAH), rendering it nonfunctional. PAH isnecessary to metabolize the amino acid phenylalanine (Phe) to the aminoacid tyrosine. When PAH activity is reduced, phenylalanine accumulatesand is converted into phenylpyruvate (also known as phenylketone). Leftuntreated, PKU can result in mental retardation, seizures and otherserious medical problems. Currently, there is no cure for the diseaseand standard of care is through management of diet, minimizing foodsthat contain high amounts of protein.

SUMMARY OF THE INVENTION

The present invention provides, among other things, methods andcompositions for the effective treatment of phenylketonurea (PKU) baseon mRNA therapy. The present invention is based, in part, on asuccessful animal study using a PKU disease model. For example, asdescribed in more detail in the examples section below, administrationof an mRNA encoding a human PAH protein, encapsulated within a liposome,resulted in efficient protein production in serum, liver and otherclinically relevant tissues in vivo. More importantly and surprisingly,treatment of PAH knockout mice, a PKU disease model, with PAH mRNA caneffectively bring down phenylalanine levels to wild type levels withinsix hours of dosing. Thus, the present inventors have demonstrated thatmRNA therapy described herein can be highly effective in treating PKU.

In one aspect, the present invention provides methods of treating PKUincluding administering to a subject in need of treatment a compositioncomprising an mRNA encoding phenylalanine hydroxylase (PAH) at aneffective dose and an administration interval such that at least onesymptom or feature of PKU is reduced in intensity, severity, orfrequency or has delayed in onset.

In another aspect, the present invention provides compositions fortreating phenylketonuria (PKU) comprising an mRNA encoding phenylalaninehydroxylase (PAH) at an effective dose amount encapsulated within aliposome.

In some embodiments, the mRNA is encapsulated within a liposome. In someembodiments, a suitable liposome comprises one or more cationic lipids,one or more non-cationic lipids, one or more cholesterol-based lipidsand one or more PEG-modified lipids.

In some embodiments, the one or more cationic lipids are selected fromthe group consisting of C12-200, MC3, DLinDMA, DLinkC2DMA, cKK-E12, ICE(Imidazol-based), HGT5000, HGT5001, DODAC, DDAB, DMRIE, DOSPA, DOGS,DODAP, DODMA and DMDMA, DODAC, DLenDMA, DMRIE, CLinDMA, CpLinDMA, DMOBA,DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA,HGT4003, and combinations thereof.

In some embodiments, the one or more cationic lipids comprise a compoundof formula I-c1-a:

or a pharmaceutically acceptable salt thereof, wherein:

-   each R² independently is hydrogen or C₁₋₃ alkyl;-   each q independently is 2 to 6;-   each R′ independently is hydrogen or C₁₋₃ alkyl;-   and each R^(L) independently is C₈₋₁₂ alkyl.

In some embodiments, the one or more cationic lipids comprise cKK-E12:

In some embodiments, the one or more non-cationic lipids are selectedfrom distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine(SOPE), or a mixture thereof.

In some embodiments, the one or more cholesterol-based lipids areselected from cholesterol, PEGylated cholesterol and DC-Chol(N,N-dimethyl-N-ethylcarboxamidocholesterol),1,4-bis(3-N-oleylamino-propyl)piperazine.

In some embodiments, the liposome further comprises one or morePEG-modified lipids. In some embodiments, the one or more PEG-modifiedlipids comprise a poly(ethylene) glycol chain of up to 5 kDa in lengthcovalently attached to a lipid with alkyl chain(s) of C₆-C₂₀ length. Insome embodiments, a PEG-modified lipid is a derivatized ceramide such asN-Octanoyl-Sphingosine-1-[Succinyl(Methoxy Polyethylene Glycol)-2000].In some embodiments, a PEG-modified or PEGylated lipid is PEGylatedcholesterol or Dimyristoylglycerol (DMG)-PEG-2K.

In some embodiments, the liposome comprises cKK-E12, DOPE, cholesterol,and DMG-PEG2K.

In some embodiments, the cationic lipid (e.g., cKK-E12) constitutesabout 30-60% (e.g., about 30-55%, about 30-50%, about 30-45%, about30-40%, about 35-50%, about 35-45%, or about 35-40%) of the liposome bymolar ratio. In some embodiments, the cationic lipid (e.g., cKK-E12)constitutes about 30%, about 35%, about 40%, about 45%, about 50%, about55%, or about 60% of the liposome by molar ratio.

In some embodiments, the ratio of cationic lipid (e.g., cKK-E12) tonon-cationic lipid (e.g., DOPE) to cholesterol-based lipid (e.g.,cholesterol) to PEGylated lipid (e.g., DMG-PEG2K) may be between about30-60:25-35:20-30:1-15, respectively. In some embodiments, the ratio ofcationic lipid (e.g., cKK-E12) to non-cationic lipid (e.g., DOPE) tocholesterol-based lipid (e.g., cholesterol) to PEGylated lipid (e.g.,DMG-PEG2K) is approximately 40:30:20:10, respectively. In someembodiments, the ratio of cationic lipid (e.g., cKK-E12) to non-cationiclipid (e.g., DOPE) to cholesterol-based lipid (e.g., cholesterol) toPEGylated lipid (e.g., DMG-PEG2K) is approximately 40:30:25:5,respectively. In some embodiments, the ratio of cationic lipid (e.g.,cKK-E12) to non-cationic lipid (e.g., DOPE) to cholesterol-based lipid(e.g., cholesterol) to PEGylated lipid (e.g., DMG-PEG2K) isapproximately 40:32:25:3, respectively. In some embodiments, the ratioof cationic lipid (e.g., cKK-E12) to non-cationic lipid (e.g., DOPE) tocholesterol-based lipid (e.g., cholesterol) to PEGylated lipid (e.g.,DMG-PEG2K) is approximately 50:25:20:5.

In some embodiments, the size of a liposome is determined by the lengthof the largest diameter of the lipososme particle. In some embodiments,a suitable liposome has a size less than about 500 nm, 400 nm, 300 nm,250 nm, 200 nm, 150 nm, 100 nm, 75 nm, or 50 nm. In some embodiments, asuitable liposome has a size less than about 100 nm, 90 nm, 80 nm, 70nm, or 60 nm.

In some embodiments, provided composition is administered intravenously.In some embodiments, provided composition is administered via pulmonarydelivery. In certain embodiments, pulmonary delivery is performed byaerosolization, inhalation, nebulization or instillation. In someembodiments, provided compositions are formulated as respirableparticles, nebulizable lipid, or inhalable dry powder.

In some embodiments, provided compositions are administered once daily,once a week, once every two weeks, twice a month, once a month. In someembodiments, provided compositions are administered once every 7 days,once every 10 days, once every 14 days, once every 28 days, or onceevery 30 days.

In some embodiments, the mRNA is administered at a dose ranging fromabout 0.1-5.0 mg/kg body weight, for example about 0.1-4.5, 0.1-4.0,0.1-3.5, 0.1-3.0, 0.1-2.5, 0.1-2.0, 0.1-1.5, 0.1-1.0, 0.1-0.5, 0.1-0.3,0.3-5.0, 0.3-4.5, 0.3-4.0, 0.3-3.5, 0.3-3.0, 0.3-2.5, 0.3-2.0, 0.3-1.5,0.3-1.0, 0.3-0.5, 0.5-5.0, 0.5-4.5, 0.5-4.0, 0.5-3.5, 0.5-3.0, 0.5-2.5,0.5-2.0, 0.5-1.5, or 0.5-1.0 mg/kg body weight. In some embodiments, themRNA is administered at a dose of or less than about 5.0, 4.5, 4.0, 3.5,3.0, 2.5, 2.0, 1.5, 1.0, 0.8, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 mg/kg bodyweight.

In some embodiments, the expression of PAH protein is detectable inliver, kidney, heart, spleen, serum, brain, skeletal muscle, lymphnodes, skin, and/or cerebrospinal fluid.

In some embodiments, administering the provided composition results inthe expression of a PAH protein level at or above about 100 ng/mg, about200 ng/mg, about 300 ng/mg, about 400 ng/mg, about 500 ng/mg, about 600ng/mg, about 700 ng/mg, about 800 ng/mg, about 900 ng/mg, about 1000ng/mg, about 1200 ng/mg or about 1400 ng/mg of total protein in theliver.

In some embodiments, the expression of the PAH protein is detectable 6,12, 18, 24, 30, 36, 42, 48, 54, 60, 66, and/or 72 hours after theadministration. In some embodiments, the expression of the PAH proteinis detectable 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, and/or 7days after the administration. In some embodiments, the expression ofthe PAH protein is detectable 1 week, 2 weeks, 3 weeks, and/or 4 weeksafter the administration. In some embodiments, the expression of the PAHprotein is detectable after a month after the administration.

In some embodiments, administering provided compositions results inincreased serum PAH protein levels. In some embodiments, administeringprovided compositions results in increased serum PAH protein levels byat least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% ascompared to baseline PAH protein level before treatment.

In some embodiments, administering provided compositions results in areduced phenylalanine level in serum as compared to baselinephenylalanine level before treatment. In some embodiments, administeringprovided compositions results in reduction of phenylalanine levels toabout 1500 μmol/L or less, about 1000 μmol/L or less, about 900 μmol/Lor less, about 800 μmol/L or less, about 700 μmol/L or less, about 600μmol/L or less, about 500 μmol/L or less, about 400 μmol/L or less,about 300 μmol/L or less, about 200 μmol/L or less, about 100 mmol/L orless or about 50 μmol/L or less in serum or plasma. In a particularembodiment, a therapeutically effective dose, when administeredregularly results in reduction of phenylalanine levels to about 120μmol/L or less in serum or plasma.

In some embodiments, administering the provided composition results inreduction of phenylalanine levels in a biological sample (e.g., a serum,plasma, or urine sample) by at least about 5%, at least about 10%, atleast about 15%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, or at least about 95% as comparedto baseline phenylalanine levels before treatment.

In some embodiments, the mRNA encoding PAH is codon optimized. In someembodiments, the codon-optimized mRNA comprises SEQ ID NO:3(corresponding to codon-optimized human PAH mRNA sequence). In someembodiments, the mRNA comprises the 5′UTR sequence of SEQ ID NO:4(corresponding to 5′ UTR sequence X). In some embodiments, the mRNAcomprises the 3′ UTR sequence of SEQ ID NO:5 (corresponding to a 3′ UTRsequence Y). In some embodiments, the mRNA comprises the 3′ UTR sequenceof SEQ ID NO:6 (corresponding to a 3′ UTR sequence Y). In someembodiments, the codon-optimized mRNA comprises SEQ ID NO:7 or SEQ IDNO:8 (corresponding to codon-optimized human PAH mRNA sequence with 5′UTR and 3′ UTR sequences).

In some embodiments, the mRNA comprises one or more modifiednucleotides. In some embodiments, the one or more modified nucleotidescomprise pseudouridine, N-1-methyl-pseudouridine, 2-aminoadenosine,2-thiothymidine, inosine, pyrrolo-yrimidine, 3-methyl adenosine,5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl -uridine,2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine,C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine,2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine,8-oxoguanosine, O(6)-methylguanine, and/or 2-thiocytidine. In someembodiments, the mRNA is unmodified.

In particular embodiments, the present invention provides compositionsfor treating phenylketonuria (PKU) including an mRNA encodingphenylalanine hydroxylase (PAH) at an effective dose amount encapsulatedwithin a liposome, wherein the mRNA comprises SEQ ID NO:3, and furtherwherein the liposome comprises cationic or non-cationic lipid,cholesterol-based lipid and PEG-modified lipid.

In particular embodiments, the present invention provides compositionsfor treating phenylketonuria (PKU) including an mRNA encodingphenylalanine hydroxylase (PAH) at an effective dose amount encapsulatedwithin a liposome, wherein the mRNA comprises SEQ ID NO:7 or SEQ IDNO:8, and further wherein the liposome comprises cationic ornon-cationic lipid, cholesterol-based lipid and PEG-modified lipid.

Other features, objects, and advantages of the present invention areapparent in the detailed description, drawings and claims that follow.It should be understood, however, that the detailed description, thedrawings, and the claims, while indicating embodiments of the presentinvention, are given by way of illustration only, not limitation.Various changes and modifications within the scope of the invention willbecome apparent to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWING

The drawings are for illustration purposes only not for limitation.

FIG. 1 shows exemplary PAH protein levels in HEK293 cells aftertransfection with provided liposomes.

FIG. 2 depicts an exemplary graph of PAH protein levels detected in theliver of wild type mice treated with provided lipid nanoparticles atvarious time points after administration.

FIG. 3 depicts an exemplary graph of PAH protein levels detected in theliver of PAH KO mice treated with provided lipid nanoparticles at 6, 12and 24 hours after administration as compared to untreated wild typemice and untreated PAH KO mice.

FIG. 4 shows an exemplary graph of serum phenylalanine levels in PAH KOmice 6, 12, and 24 hours after treatment with provided lipidnanoparticles as compared to untreated wild type mice and untreated PAHKO mice.

FIGS. 5A-5I depicts in situ detection of human PAH mRNA in liver tissuefrom mice (A) 30 minutes, (B) 3 hours, (C) 6 hours, (D) 12 hours, (E) 24hours, (F) 48 hours, (G) 72 hours or (H) 7 days after treatment with 1.0mg/kg of hPAH mRNA-loaded cKK-E12-based lipid nanoparticles, or fromuntreated mice (I).

FIG. 6 depicts an exemplary graph of human PAH protein levels detectedin the liver of PAH knock-out mice treated with a single dose of 0.25mg/kg, 0.5 mg/kg, 0.75 mg/kg or 1.0 mg/kg of hPAH mRNA-loadedcKK-E12-based lipid nanoparticles, or saline.

FIG. 7 depicts an exemplary graph of phenylalanine levels detected inthe serum of PAH knock-out mice prior to treatment and followingtreatment with a single dose of 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg or 1.0mg/kg of hPAH mRNA-loaded cKK-E12-based lipid nanoparticles, or withsaline.

FIG. 8 depicts an exemplary graph of human PAH protein levels detectedin the liver of PAH knock-out mice treated with 0.5 mg/kg or 1.0 mg/kgof hPAH mRNA-loaded cKK-E12-based lipid nanoparticles once per week forone month, or with 1.0 mg/kg of hPAH mRNA-loaded cKK-E12-based lipidnanoparticles every other week for one month, or with saline.

FIG. 9 depicts an exemplary graph of phenylalanine levels detected inthe serum of PAH knock-out mice prior to treatment and followingtreatment with 0.5 mg/kg or 1.0 mg/kg of hPAH mRNA-loaded cKK-E12-basedlipid nanoparticles once per week for one month, or with 1.0 mg/kg ofhPAH mRNA-loaded cKK-E12-based lipid nanoparticles every other week forone month, or with saline.

DEFINITIONS

In order for the present invention to be more readily understood,certain terms are first defined below. Additional definitions for thefollowing terms and other terms are set forth throughout thespecification. The publications and other reference materials referencedherein to describe the background of the invention and to provideadditional detail regarding its practice are hereby incorporated byreference.

Alkyl: As used herein, “alkyl” refers to a radical of a straight-chainor branched saturated hydrocarbon group having from 1 to 15 carbon atoms(“C₁₋₁₅ alkyl”). In some embodiments, an alkyl group has 1 to 3 carbonatoms (“C₁₋₃ alkyl”). Examples of C₁₋₃ alkyl groups include methyl (C₁),ethyl (C₂), n-propyl (C₃), and isopropyl (C₃). In some embodiments, analkyl group has 8 to 12 carbon atoms (“C₈₋₁₂ alkyl”). Examples of C₈₋₁₂alkyl groups include, without limitation, n-octyl (C₈), n-nonyl (C₉),n-decyl (C₁₀), n-undecyl (C₁₁), n-dodecyl (C₁₂) and the like. The prefix“n-” (normal) refers to unbranched alkyl groups. For example, n-C₈ alkylrefers to —(CH₂)₇CH₃, n-C₁₀ alkyl refers to —(CH₂)₉CH₃, etc.

Amino acid: As used herein, term “amino acid,” in its broadest sense,refers to any compound and/or substance that can be incorporated into apolypeptide chain. In some embodiments, an amino acid has the generalstructure H₂N—C(H)(R)—COOH. In some embodiments, an amino acid is anaturally occurring amino acid. In some embodiments, an amino acid is asynthetic amino acid; in some embodiments, an amino acid is a d-aminoacid; in some embodiments, an amino acid is an 1-amino acid. “Standardamino acid” refers to any of the twenty standard 1-amino acids commonlyfound in naturally occurring peptides. “Nonstandard amino acid” refersto any amino acid, other than the standard amino acids, regardless ofwhether it is prepared synthetically or obtained from a natural source.As used herein, “synthetic amino acid” encompasses chemically modifiedamino acids, including but not limited to salts, amino acid derivatives(such as amides), and/or substitutions. Amino acids, including carboxy-and/or amino-terminal amino acids in peptides, can be modified bymethylation, amidation, acetylation, protecting groups, and/orsubstitution with other chemical groups that can change the peptide'scirculating half-life without adversely affecting their activity. Aminoacids may participate in a disulfide bond. Amino acids may comprise oneor posttranslational modifications, such as association with one or morechemical entities (e.g., methyl groups, acetate groups, acetyl groups,phosphate groups, formyl moieties, isoprenoid groups, sulfate groups,polyethylene glycol moieties, lipid moieties, carbohydrate moieties,biotin moieties, etc.). The term “amino acid” is used interchangeablywith “amino acid residue,” and may refer to a free amino acid and/or toan amino acid residue of a peptide. It will be apparent from the contextin which the term is used whether it refers to a free amino acid or aresidue of a peptide.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans, at anystage of development. In some embodiments, “animal” refers to non-humananimals, at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). Insome embodiments, animals include, but are not limited to, mammals,birds, reptiles, amphibians, fish, insects, and/or worms. In someembodiments, an animal may be a transgenic animal,genetically-engineered animal, and/or a clone.

Approximately or about: As used herein, the term “approximately” or“about,” as applied to one or more values of interest, refers to a valuethat is similar to a stated reference value. In certain embodiments, theterm “approximately” or “about” refers to a range of values that fallwithin 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greaterthan or less than) of the stated reference value unless otherwise statedor otherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Biologically active: As used herein, the phrase “biologically active”refers to a characteristic of any agent that has activity in abiological system, and particularly in an organism. For instance, anagent that, when administered to an organism, has a biological effect onthat organism, is considered to be biologically active.

Delivery: As used herein, the term “delivery” encompasses both local andsystemic delivery. For example, delivery of mRNA encompasses situationsin which an mRNA is delivered to a target tissue and the encoded proteinis expressed and retained within the target tissue (also referred to as“local distribution” or “local delivery”), and situations in which anmRNA is delivered to a target tissue and the encoded protein isexpressed and secreted into patient's circulation system (e.g., serum)and systematically distributed and taken up by other tissues (alsoreferred to as “systemic distribution” or “systemic delivery).

Expression: As used herein, “expression” of a nucleic acid sequencerefers to translation of an mRNA into a polypeptide, assemble multiplepolypeptides into an intact protein (e.g., enzyme) and/orpost-translational modification of a polypeptide or fully assembledprotein (e.g., enzyme). In this application, the terms “expression” and“production,” and grammatical equivalent, are used inter-changeably.

Functional: As used herein, a “functional” biological molecule is abiological molecule in a form in which it exhibits a property and/oractivity by which it is characterized.

Half-life: As used herein, the term “half-life” is the time required fora quantity such as nucleic acid or protein concentration or activity tofall to half of its value as measured at the beginning of a time period.

Improve, increase, or reduce: As used herein, the terms “improve,”“increase” or “reduce,” or grammatical equivalents, indicate values thatare relative to a baseline measurement, such as a measurement in thesame individual prior to initiation of the treatment described herein,or a measurement in a control subject (or multiple control subject) inthe absence of the treatment described herein. A “control subject” is asubject afflicted with the same form of disease as the subject beingtreated, who is about the same age as the subject being treated.

In Vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, etc., rather than within a multi-cellularorganism.

In Vivo: As used herein, the term “in vivo” refers to events that occurwithin a multi-cellular organism, such as a human and a non-humananimal. In the context of cell-based systems, the term may be used torefer to events that occur within a living cell (as opposed to, forexample, in vitro systems).

Isolated: As used herein, the term “isolated” refers to a substanceand/or entity that has been (1) separated from at least some of thecomponents with which it was associated when initially produced (whetherin nature and/or in an experimental setting), and/or (2) produced,prepared, and/or manufactured by the hand of man. Isolated substancesand/or entities may be separated from about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98%, about 99%, or more than about 99% of the other componentswith which they were initially associated. In some embodiments, isolatedagents are about 80%, about 85%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,or more than about 99% pure. As used herein, a substance is “pure” if itis substantially free of other components. As used herein, calculationof percent purity of isolated substances and/or entities should notinclude excipients (e.g., buffer, solvent, water, etc.).

Local distribution or delivery: As used herein, the terms “localdistribution,” “local delivery,” or grammatical equivalent, refer totissue specific delivery or distribution. Typically, local distributionor delivery requires a protein (e.g., enzyme) encoded by mRNAs betranslated and expressed intracellularly or with limited secretion thatavoids entering the patient's circulation system.

messenger RNA (mRNA): As used herein, the term “messenger RNA (mRNA)”refers to a polynucleotide that encodes at least one polypeptide. mRNAas used herein encompasses both modified and unmodified RNA. mRNA maycontain one or more coding and non-coding regions. mRNA can be purifiedfrom natural sources, produced using recombinant expression systems andoptionally purified, chemically synthesized, etc. Where appropriate,e.g., in the case of chemically synthesized molecules, mRNA can comprisenucleoside analogs such as analogs having chemically modified bases orsugars, backbone modifications, etc. An mRNA sequence is presented inthe 5′ to 3′ direction unless otherwise indicated. In some embodiments,an mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine,cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine,2-thiothymidine, inosine, pyrrolo -pyrimidine, 3-methyl adenosine,5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl -uridine,2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine,C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine,2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine,8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemicallymodified bases; biologically modified bases (e.g., methylated bases);intercalated bases; modified sugars (e.g., 2′-fluororibose, ribose,2′-deoxyribose, arabinose, and hexose); and/or modified phosphate groups(e.g., phosphorothioates and 5′-N-phosphoramidite linkages).

Nucleic acid: As used herein, the term “nucleic acid,” in its broadestsense, refers to any compound and/or substance that is or can beincorporated into a polynucleotide chain. In some embodiments, a nucleicacid is a compound and/or substance that is or can be incorporated intoa polynucleotide chain via a phosphodiester linkage. In someembodiments, “nucleic acid” refers to individual nucleic acid residues(e.g., nucleotides and/or nucleosides). In some embodiments, “nucleicacid” refers to a polynucleotide chain comprising individual nucleicacid residues. In some embodiments, “nucleic acid” encompasses RNA aswell as single and/or double-stranded DNA and/or cDNA.

Patient: As used herein, the term “patient” or “subject” refers to anyorganism to which a provided composition may be administered, e.g., forexperimental, diagnostic, prophylactic, cosmetic, and/or therapeuticpurposes. Typical patients include animals (e.g., mammals such as mice,rats, rabbits, non-human primates, and/or humans). In some embodiments,a patient is a human. A human includes pre and post natal forms.

Pharmaceutically acceptable: The term “pharmaceutically acceptable” asused herein, refers to substances that, within the scope of soundmedical judgment, are suitable for use in contact with the tissues ofhuman beings and animals without excessive toxicity, irritation,allergic response, or other problem or complication, commensurate with areasonable benefit/risk ratio.

Pharmaceutically acceptable salt: Pharmaceutically acceptable salts arewell known in the art. For example, S. M. Berge et al., describespharmaceutically acceptable salts in detail in J. PharmaceuticalSciences (1977) 66:1-19. Pharmaceutically acceptable salts of thecompounds of this invention include those derived from suitableinorganic and organic acids and bases. Examples of pharmaceuticallyacceptable, nontoxic acid addition salts are salts of an amino groupformed with inorganic acids such as hydrochloric acid, hydrobromic acid,phosphoric acid, sulfuric acid and perchloric acid or with organic acidssuch as acetic acid, oxalic acid, maleic acid, tartaric acid, citricacid, succinic acid or malonic acid or by using other methods used inthe art such as ion exchange. Other pharmaceutically acceptable saltsinclude adipate, alginate, ascorbate, aspartate, benzenesulfonate,benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate,citrate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate,gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p -toluenesulfonate, undecanoate, valerate salts, and thelike. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N⁺(C₁₋₄ alkyl)₄ salts. Representativealkali or alkaline earth metal salts include sodium, lithium, potassium,calcium, magnesium, and the like. Further pharmaceutically acceptablesalts include, when appropriate, nontoxic ammonium. quaternary ammonium,and amine cations formed using counterions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, sulfonate and aryl sulfonate.Further pharmaceutically acceptable salts include salts formed from thequaternization of an amine using an appropriate electrophile, e.g., analkyl halide, to form a quarternized alkylated amino salt.

Systemic distribution or delivery: As used herein, the terms “systemicdistribution,” “systemic delivery,” or grammatical equivalent, refer toa delivery or distribution mechanism or approach that affect the entirebody or an entire organism. Typically, systemic distribution or deliveryis accomplished via body's circulation system, e.g., blood stream.Compared to the definition of “local distribution or delivery.”

Subject: As used herein, the term “subject” refers to a human or anynon-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine,sheep, horse or primate). A human includes pre- and post-natal forms. Inmany embodiments, a subject is a human being. A subject can be apatient, which refers to a human presenting to a medical provider fordiagnosis or treatment of a disease. The term “subject” is used hereininterchangeably with “individual” or “patient.” A subject can beafflicted with or is susceptible to a disease or disorder but may or maynot display symptoms of the disease or disorder.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Target tissues: As used herein, the term “target tissues” refers to anytissue that is affected by a disease to be treated. In some embodiments,target tissues include those tissues that display disease-associatedpathology, symptom, or feature.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” of a therapeutic agent means anamount that is sufficient, when administered to a subject suffering fromor susceptible to a disease, disorder, and/or condition, to treat,diagnose, prevent, and/or delay the onset of the symptom(s) of thedisease, disorder, and/or condition. It will be appreciated by those ofordinary skill in the art that a therapeutically effective amount istypically administered via a dosing regimen comprising at least one unitdose.

Treating: As used herein, the term “treat,” “treatment,” or “treating”refers to any method used to partially or completely alleviate,ameliorate, relieve, inhibit, prevent, delay onset of, reduce severityof and/or reduce incidence of one or more symptoms or features of aparticular disease, disorder, and/or condition. Treatment may beadministered to a subject who does not exhibit signs of a disease and/orexhibits only early signs of the disease for the purpose of decreasingthe risk of developing pathology associated with the disease.

DETAILED DESCRIPTION

The present invention provides, among other things, methods andcompositions for treating phenylketonuria (PKU) based on mRNA therapy.In particular, the present invention provides methods for treating PKUby administering to a subject in need of treatment a compositioncomprising an mRNA encoding phenylalanine hydroxylase (PAH) at aneffective dose and an administration interval such that at least onesymptom or feature of PKU is reduced in intensity, severity, orfrequency or has delayed in onset. In some embodiments, the mRNA isencapsulated within a liposome. As used herein, the term “liposome”refers to any lamellar, multilamellar, or solid lipid nanoparticlevesicle. Typically, a liposome as used herein can be formed by mixingone or more lipids or by mixing one or more lipids and polymer(s). Thus,the term “liposome” as used herein encompasses both lipid and polymerbased nanoparticles. In some embodiments, a liposome suitable for thepresent invention contains cationic or non-cationic lipid(s),cholesterol-based lipid(s) and PEG-modified lipid(s).

Phenylketonuria (PKU)

The present invention may be used to treat a subject who is sufferingfrom or susceptible to Phenylketonuria (PKU). PKU is an autosomalrecessive metabolic genetic disorder characterized by a mutation in thegene for the hepatic enzyme phenylalanine hydroxylase (PAH), renderingit nonfunctional. PAH is necessary to metabolize the amino acidphenylalanine (Phe) to the amino acid tyrosine. When PAH activity isreduced, phenylalanine accumulates and is converted into phenylpyruvate(also known as phenylketone) which can be detected in the urine.

Phenylalanine is a large, neutral amino acid (LNAA). LNAAs compete fortransport across the blood-brain barrier (BBB) via the large neutralamino acid transporter (LNAAT). Excess Phe in the blood saturates thetransporter and tends to decrease the levels of other LNAAs in thebrain. Because several of these other amino acids are necessary forprotein and neurotransmitter synthesis, Phe buildup hinders thedevelopment of the brain, and can cause mental retardation.

In addition to hindered brain development, the disease can presentclinically with a variety of symptoms including seizures, albinismhyperactivity, stunted growth, skin rashes (eczema), microcephaly,and/or a “musty” odor to the baby's sweat and urine, due tophenylacetate, one of the ketones produced). Untreated children aretypically normal at birth, but have delayed mental and social skills,have a head size significantly below normal, and often demonstrateprogressive impairment of cerebral function. As the child grows anddevelops, additional symptoms including hyperactivity, jerking movementsof the arms or legs, EEG abnormalities, skin rashes, tremors, seizures,and severe learning disabilities tend to develop. However, PKU iscommonly included in the routine newborn screening panel of mostcountries that is typically performed 2-7 days after birth.

If PKU is diagnosed early enough, an affected newborn can grow up withrelatively normal brain development, but only by managing andcontrolling Phe levels through diet, or a combination of diet andmedication. All PKU patients must adhere to a special diet low in Phefor optimal brain development. The diet requires severely restricting oreliminating foods high in Phe, such as meat, chicken, fish, eggs, nuts,cheese, legumes, milk and other dairy products. Starchy foods, such aspotatoes, bread, pasta, and corn, must be monitored. Infants may stillbe breastfed to provide all of the benefits of breastmilk, but thequantity must also be monitored and supplementation for missingnutrients will be required. The sweetener aspartame, present in manydiet foods and soft drinks, must also be avoided, as aspartame containsphenylalanine.

Throughout life, patients can use supplementary infant formulas, pillsor specially formulated foods to acquire amino acids and other necessarynutrients that would otherwise be deficient in a low-phenylalanine diet.Some Phe is required for the synthesis of many proteins and is requiredfor appropriate growth, but levels of it must be strictly controlled inPKU patients. Additionally, PKU patients must take supplements oftyrosine, which is normally derived from phenylalanine. Othersupplements can include fish oil, to replace the long chain fatty acidsmissing from a standard Phe-free diet and improve neurologicaldevelopment and iron or carnitine. Another potential therapy for PKU istetrahydrobiopterin (BH4), a cofactor for the oxidation of Phe that canreduce blood levels of Phe in certain patients. Patients who respond toBH4 therapy may also be able to increase the amount of natural proteinthat they can eat.

Phenylalanine Hydroxylase (PAH)

In some embodiments, the present invention provides methods andcompositions for delivering mRNA encoding PAH to a subject for thetreatment of phenylketonuria (PKU). A suitable PAH mRNA encodes any fulllength, fragment or portion of a PAH protein which can be substitutedfor naturally-occurring PAH protein activity and/or reduce theintensity, severity, and/or frequency of one or more symptoms associatedwith PKU.

In some embodiments, a suitable mRNA sequence for the present inventioncomprises an mRNA sequence encoding human PAH protein. Thenaturally-occurring human PAH mRNA and the corresponding amino acidsequence are shown in Table 1:

TABLE 1 Human PAH HumanCAGCUGGGGGUAAGGGGGGCGGAUUAUUCAUAUAAUUGUUAUACCAGACGG PAHUCGCAGGCUUAGUCCAAUUGCAGAGAACUCGCUUCCCAGGCUUCUGAGAGUC (mRNA)CCGGAAGUGCCUAAACCUGUCUAAUCGACGGGGCUUGGGUGGCCCGUCGCUCCCUGGCUUCUUCCCUUUACCCAGGGCGGGCAGCGAAGUGGUGCCUCCUGCGUCCCCCACACCCUCCCUCAGCCCCUCCCCUCCGGCCCGUCCUGGGCAGGUGACCUGGAGCAUCCGGCAGGCUGCCCUGGCCUCCUGCGUCAGGACAAGCCCACGAGGGGCGUUACUGUGCGGAGAUGCACCACGCAAGAGACACCCUUUGUAACUCUCUUCUCCUCCCUAGUGCGAGGUUAAAACCUUCAGCCCCACGUGCUGUUUGCAAACCUGCCUGUACCUGAGGCCCUAAAAAGCCAGAGACCUCACUCCCGGGGAGCCAGCAUGUCCACUGCGGUCCUGGAAAACCCAGGCUUGGGCAGGAAACUCUCUGACUUUGGACAGGAAACAAGCUAUAUUGAAGACAACUGCAAUCAAAAUGGUGCCAUAUCACUGAUCUUCUCACUCAAAGAAGAAGUUGGUGCAUUGGCCAAAGUAUUGCGCUUAUUUGAGGAGAAUGAUGUAAACCUGACCCACAUUGAAUCUAGACCUUCUCGUUUAAAGAAAGAUGAGUAUGAAUUUUUCACCCAUUUGGAUAAACGUAGCCUGCCUGCUCUGACAAACAUCAUCAAGAUCUUGAGGCAUGACAUUGGUGCCACUGUCCAUGAGCUUUCACGAGAUAAGAAGAAAGACACAGUGCCCUGGUUCCCAAGAACCAUUCAAGAGCUGGACAGAUUUGCCAAUCAGAUUCUCAGCUAUGGAGCGGAACUGGAUGCUGACCACCCUGGUUUUAAAGAUCCUGUGUACCGUGCAAGACGGAAGCAGUUUGCUGACAUUGCCUACAACUACCGCCAUGGGCAGCCCAUCCCUCGAGUGGAAUACAUGGAGGAAGAAAAGAAAACAUGGGGCACAGUGUUCAAGACUCUGAAGUCCUUGUAUAAAACCCAUGCUUGCUAUGAGUACAAUCACAUUUUUCCACUUCUUGAAAAGUACUGUGGCUUCCAUGAAGAUAACAUUCCCCAGCUGGAAGACGUUUCUCAAUUCCUGCAGACUUGCACUGGUUUCCGCCUCCGACCUGUGGCUGGCCUGCUUUCCUCUCGGGAUUUCUUGGGUGGCCUGGCCUUCCGAGUCUUCCACUGCACACAGUACAUCAGACAUGGAUCCAAGCCCAUGUAUACCCCCGAACCUGACAUCUGCCAUGAGCUGUUGGGACAUGUGCCCUUGUUUUCAGAUCGCAGCUUUGCCCAGUUUUCCCAGGAAAUUGGCCUUGCCUCUCUGGGUGCACCUGAUGAAUACAUUGAAAAGCUCGCCACAAUUUACUGGUUUACUGUGGAGUUUGGGCUCUGCAAACAAGGAGACUCCAUAAAGGCAUAUGGUGCUGGGCUCCUGUCAUCCUUUGGUGAAUUACAGUACUGCUUAUCAGAGAAGCCAAAGCUUCUCCCCCUGGAGCUGGAGAAGACAGCCAUCCAAAAUUACACUGUCACGGAGUUCCAGCCCCUGUAUUACGUGGCAGAGAGUUUUAAUGAUGCCAAGGAGAAAGUAAGGAACUUUGCUGCCACAAUACCUCGGCCCUUCUCAGUUCGCUACGACCCAUACACCCAAAGGAUUGAGGUCUUGGACAAUACCCAGCAGCUUAAGAUUUUGGCUGAUUCCAUUAACAGUGAAAUUGGAAUCCUUUGCAGUGCCCUCCAGAAAAUAAAGUAAAGCCAUGGACAGAAUGUGGUCUGUCAGCUGUGAAUCUGUUGAUGGAGAUCCAACUAUUUCUUUCAUCAGAAAAAGUCCGAAAAGCAAACCUUAAUUUGAAAUAACAGCCUUAAAUCCUUUACAAGAUGGAGAAACAACAAAUAAGUCAAAAUAAUCUGAAAUGACAGGAUAUGAGUACAUACUCAAGAGCAUAAUGGUAAAUCUUUUGGGGUCAUCUUUGAUUUAGAGAUGAUAAUCCCAUACUCUCAAUUGAGUUAAAUCAGUAAUCUGUCGCAUUUCAUCAAGAUUAAUUAAAAUUUGGGACCUGCUUCAUUCAAGCUUCAUAUAUGCUUUGCAGAGAACUCAUAAAGGAGCAUAUAAGGCUAAAUGUAAAACACAAGACUGUCAUUAGAAUUGAAUUAUUGGGCUUAAUAUAAAUCGUAACCUAUGAAGUUUAUUUUCUAUUUUAGUUAACUAUGAUUCCAAUUACUACUUUGUUAUUGUACCUAAGUAAAUUUUCUUUAGGUCAGAAGCCCAUUAAAAUAGUUACAAGCAUUGAACUUCUUUAGUAUUAUAUUAAUAUAAAAACAUUUUUGUAUGUUUUAUUGUAAUCAUAAAUACUGCUGUAUAAGGUAAUAAAACUCUGCACCUAAUCCCCAUAACUUCCAGUAUCAUUUUCCAAUUAAUUAUCAAGUCUGUUUUGGGAAACACUUUGAGGACAUUUAUGAUGCAGCAGAUGUUGACUAAAGGCUUGGUUGGUAGAUAUUCAGGAAAUGUUCACUGAAUAAAUAAGUAAAUACAUUAUUGAAAAGCAAAUCUGUAUAAAUGUGAAAUUUUUAUUUGUAUUAGUAAUAAAACAUUAGUAGUUUA (SEQ ID NO: 1) HumanMSTAVLENPGLGRKLSDFGQETSYIEDNCNQNGAISLIFSLKEEVGALAKVLRLFEE PAHNDVNLTHIESRPSRLKKDEYEFFTHLDKRSLPALTNIIKILRHDIGATVHELSRDKKK (AminoDTVPWFPRTIQELDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRH Acid Seq.)GQPIPRVEYMEEEKKTWGTVFKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEKLATIYWFTVEFGLCKQGDSIKAYGAGLLSSFGELQYCLSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAATIPRPFSVRYDPYTQRIEVLDNTQQLKILADSINSEIGILCSALQKIK (SEQ ID NO: 2)

In some embodiments, a suitable mRNA is a wild-type hPAH mRNA sequence(SEQ ID NO:1). In some embodiments, a suitable mRNA may be a codonoptimized hPAH mRNA sequence, such as the sequence shown below:

(SEQ ID NO: 3) AUGAGCACCGCCGUGCUGGAGAACCCCGGCCUGGGCCGCAAGCUGAGCGACUUCGGCCAGGAGACCAGCUACAUCGAGGACAACUGCAACCAGAACGGCGCCAUCAGCCUGAUCUUCAGCCUGAAGGAGGAGGUGGGCGCCCUGGCCAAGGUGCUGCGCCUGUUCGAGGAGAACGACGUGAACCUGACCCACAUCGAGAGCCGCCCCAGCCGCCUGAAGAAGGACGAGUACGAGUUCUUCACCCACCUGGACAAGCGCAGCCUGCCCGCCCUGACCAACAUCAUCAAGAUCCUGCGCCACGACAUCGGCGCCACCGUGCACGAGCUGAGCCGCGACAAGAAGAAGGACACCGUGCCCUGGUUCCCCCGCACCAUCCAGGAGCUGGACCGCUUCGCCAACCAGAUCCUGAGCUACGGCGCCGAGCUGGACGCCGACCACCCCGGCUUCAAGGACCCCGUGUACCGCGCCCGCCGCAAGCAGUUCGCCGACAUCGCCUACAACUACCGCCACGGCCAGCCCAUCCCCCGCGUGGAGUACAUGGAGGAGGAGAAGAAGACCUGGGGCACCGUGUUCAAGACCCUGAAGAGCCUGUACAAGACCCACGCCUGCUACGAGUACAACCACAUCUUCCCCCUGCUGGAGAAGUACUGCGGCUUCCACGAGGACAACAUCCCCCAGCUGGAGGACGUGAGCCAGUUCCUGCAGACCUGCACCGGCUUCCGCCUGCGCCCCGUGGCCGGCCUGCUGAGCAGCCGCGACUUCCUGGGCGGCCUGGCCUUCCGCGUGUUCCACUGCACCCAGUACAUCCGCCACGGCAGCAAGCCCAUGUACACCCCCGAGCCCGACAUCUGCCACGAGCUGCUGGGCCACGUGCCCCUGUUCAGCGACCGCAGCUUCGCCCAGUUCAGCCAGGAGAUCGGCCUGGCCAGCCUGGGCGCCCCCGACGAGUACAUCGAGAAGCUGGCCACCAUCUACUGGUUCACCGUGGAGUUCGGCCUGUGCAAGCAGGGCGACAGCAUCAAGGCCUACGGCGCCGGCCUGCUGAGCAGCUUCGGCGAGCUGCAGUACUGCCUGAGCGAGAAGCCCAAGCUGCUGCCCCUGGAGCUGGAGAAGACCGCCAUCCAGAACUACACCGUGACCGAGUUCCAGCCCCUGUACUACGUGGCCGAGAGCUUCAACGACGCCAAGGAGAAGGUGCGCAACUUCGCCGCCACCAUCCCCCGCCCCUUCAGCGUGCGCUACGACCCCUACACCCAGCGCAUCGAGGUGCUGGACAACACCCAGCAGCUGAAGAUCCUGGCCGACAGCAUCAACAGCGAGAUCGGCAUCCUGUGCAGCGCCCUGCAGAAG AUCAAGUAA

Additional exemplary mRNA sequences are described in the Examplessection, such as, SEQ ID NO:7 and SEQ ID NO:8, both of which include 5′and 3′ untranslated regions framing a codon optimized mRNA sequence.

In some embodiments, a suitable mRNA sequence may be an mRNA sequencethat encodes a homolog or an analog of human PAH. As used herein, ahomologue or an analogue of human PAH protein may be a modified humanPAH protein containing one or more amino acid substitutions, deletions,and/or insertions as compared to a wild-type or naturally-occurringhuman PAH protein while retaining substantial PAH protein activity. Insome embodiments, an mRNA suitable for the present invention encodes anamino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous toSEQ ID NO:2. In some embodiments, an mRNA suitable for the presentinvention encodes a protein substantially identical to human PAHprotein. In some embodiments, an mRNA suitable for the present inventionencodes an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moreidentical to SEQ ID NO:2. In some embodiments, an mRNA suitable for thepresent invention encodes a fragment or a portion of human PAH protein.In some embodiments, an mRNA suitable for the present invention encodesa fragment or a portion of human PAH protein, wherein the fragment orportion of the protein still maintains PAH activity similar to that ofthe wild-type protein. In some embodiments, an mRNA suitable for thepresent invention has a nucleotide sequence at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO:7 or SEQ ID NO:8.

In some embodiments, a suitable mRNA encodes a fusion protein comprisinga full length, fragment or portion of a PAH protein fused to anotherprotein (e.g., an N or C terminal fusion). In some embodiments, theprotein fused to the mRNA encoding a full length, fragment or portion ofa PAH protein encodes a signal or a cellular targeting sequence.

Delivery Vehicles

According to the present invention, mRNA encoding a PAH protein (e.g., afull length, fragment or portion of a PAH protein) as described hereinmay be delivered as naked RNA (unpackaged) or via delivery vehicles. Asused herein, the terms “delivery vehicle,” “transfer vehicle,”“Nanoparticle” or grammatical equivalent, are used interchangeably.

In some embodiments, mRNAs encoding a PAH protein may be delivered via asingle delivery vehicle. In some embodiments, mRNAs encoding a PAHprotein may be delivered via one or more delivery vehicles each of adifferent composition. According to various embodiments, suitabledelivery vehicles include, but are not limited to polymer basedcarriers, such as polyethyleneimine (PEI), lipid nanoparticles andliposomes, nanoliposomes, ceramide-containing nanoliposomes,proteoliposomes, both natural and synthetically-derived exosomes,natural, synthetic and semi-synthetic lamellar bodies, nanoparticulates,calcium phosphor-silicate nanoparticulates, calcium phosphatenanoparticulates, silicon dioxide nanoparticulates, nanocrystallineparticulates, semiconductor nanoparticulates, poly(D-arginine),sol-gels, nanodendrimers, starch-based delivery systems, micelles,emulsions, niosomes, multi-domain-block polymers (vinyl polymers,polypropyl acrylic acid polymers, dynamic polyconjugates), dry powderformulations, plasmids, viruses, calcium phosphate nucleotides,aptamers, peptides and other vectorial tags.

Liposomal Delivery Vehicles

In some embodiments, a suitable delivery vehicle is a liposomal deliveryvehicle, e.g., a lipid nanoparticle. As used herein, liposomal deliveryvehicles, e.g., lipid nanoparticles, are usually characterized asmicroscopic vesicles having an interior aqua space sequestered from anouter medium by a membrane of one or more bilayers. Bilayer membranes ofliposomes are typically formed by amphiphilic molecules, such as lipidsof synthetic or natural origin that comprise spatially separatedhydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16:307-321, 1998). Bilayer membranes of the liposomes can also be formed byamphophilic polymers and surfactants (e.g., polymerosomes, niosomes,etc.). In the context of the present invention, a liposomal deliveryvehicle typically serves to transport a desired mRNA to a target cell ortissue. The process of incorporation of a desired mRNA into a liposomeis often referred to as “loading”. Exemplary methods are described inLasic, et al., FEBS Lett., 312: 255-258, 1992, which is incorporatedherein by reference. The liposome-incorporated nucleic acids may becompletely or partially located in the interior space of the liposome,within the bilayer membrane of the liposome, or associated with theexterior surface of the liposome membrane. The incorporation of anucleic acid into liposomes is also referred to herein as“encapsulation” wherein the nucleic acid is entirely contained withinthe interior space of the liposome. The purpose of incorporating a mRNAinto a transfer vehicle, such as a liposome, is often to protect thenucleic acid from an environment which may contain enzymes or chemicalsthat degrade nucleic acids and/or systems or receptors that cause therapid excretion of the nucleic acids. Accordingly, in some embodiments,a suitable delivery vehicle is capable of enhancing the stability of themRNA contained therein and/or facilitate the delivery of mRNA to thetarget cell or tissue.

Cationic Lipids

In some embodiments, liposomes may comprise one or more cationic lipids.As used herein, the phrase “cationic lipid” refers to any of a number oflipid species that have a net positive charge at a selected pH, such asphysiological pH. Several cationic lipids have been described in theliterature, many of which are commercially available. Particularlysuitable cationic lipids for use in the compositions and methods of theinvention include those described in international patent publicationsWO 2010/053572 (and particularly, CI 2-200 described at paragraph[00225]) and WO 2012/170930, both of which are incorporated herein byreference. In certain embodiments, the compositions and methods of theinvention employ a lipid nanoparticles comprising an ionizable cationiclipid described in U.S. provisional patent application 61/617,468, filedMar. 29, 2012 (incorporated herein by reference), such as, e.g,(15Z,18Z)-N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine(HGT5000),(15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine(HGT5001), and(15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine(HGT5002).

In some embodiments, provided liposomes include a cationic lipiddescribed in WO 2013063468 and in U.S. provisional application entitled“Lipid Formulations for Delivery of Messenger RNA” filed concurrentlywith the present application on even date, both of which areincorporated by reference herein. In some embodiments, a cationic lipidcomprises a compound of formula I-c1-a:

or a pharmaceutically acceptable salt thereof, wherein:

-   each R² independently is hydrogen or C₁₋₃ alkyl;-   each q independently is 2 to 6;-   each R′ independently is hydrogen or C₁₋₃ alkyl;-   and each R^(L) independently is C₈₋₁₂ alkyl.

In some embodiments, each R² independently is hydrogen, methyl or ethyl.In some embodiments, each R² independently is hydrogen or methyl. Insome embodiments, each R² is hydrogen.

In some embodiments, each q independently is 3 to 6. In someembodiments, each q independently is 3 to 5. In some embodiments, each qis 4.

In some embodiments, each R′ independently is hydrogen, methyl or ethyl.In some embodiments, each R′ independently is hydrogen or methyl. Insome embodiments, each R′ independently is hydrogen.

In some embodiments, each R^(L) independently is C₈₋₁₂ alkyl. In someembodiments, each R^(L) independently is n-C₈₋₁₂ alkyl. In someembodiments, each R^(L) independently is C₉₋₁₁ alkyl. In someembodiments, each R^(L) independently is n-C₉₋₁₁ alkyl. In someembodiments, each R^(L) independently is C₁₀ alkyl. In some embodiments,each R^(L) independently is n-C₁₀ alkyl.

In some embodiments, each R² independently is hydrogen or methyl; each qindependently is 3 to 5; each R′ independently is hydrogen or methyl;and each R^(L) independently is C₈₋₁₂ alkyl.

In some embodiments, each R² is hydrogen; each q independently is 3 to5; each R′ is hydrogen; and each R^(L) independently is C₈₋₁₂ alkyl.

In some embodiments, each R² is hydrogen; each q is 4; each R′ ishydrogen; and each R^(L) independently is C₈₋₁₂ alkyl.

In some embodiments, a cationic lipid comprises a compound of formulaI-g:

or a pharmaceutically acceptable salt thereof, wherein each R^(L)independently is C₈₋₁₂ alkyl. In some embodiments, each R^(L)independently is n-C₈₋₁₂ alkyl. In some embodiments, each R^(L)independently is C₉₋₁₁ alkyl. In some embodiments, each R^(L)independently is n-C₉₋₁₁ alkyl. In some embodiments, each R^(L)independently is C₁₀ alkyl. In some embodiments, each R^(L) is n-C₁₀alkyl.

In particular embodiments, provided liposomes include a cationic lipidcKK-E12, or(3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione).Structure of cKK-E12 is shown below:

As described in the Examples section below, the present inventorsobserved that liposomes based on this particular class of cationiclipids, such as, those having a structure of formula I-c1-a or formulaI-g described herein (e.g., cKK-E12) are unexpectedly effective indelivering mRNA and producing encoded protein in vivo. Although mRNAencoding PAH protein is used as an example in this application, it iscontemplated that this class of cationic lipids having a structure offormula I-c1-a or formula I-g described herein (e.g., cKK-E12) can beuseful in delivering any mRNA for highly efficient and sustainedproduction of protein (e.g., therapeutic protein) in vivo. For example,cationic lipids having a structure of formula I-c1-a or formula I-gdescribed herein (e.g., cKK-E12) can be used to deliver an mRNA thatencodes one or more naturally occurring peptides or one or more modifiedor non-natural peptides. In some embodiments, cationic lipids having astructure of formula I-c1-a or formula I-g described herein (e.g.,cKK-E12) can be used to deliver an mRNA that encodes an intracellularprotein including, but not limited to, a cytosolic protein (e.g., achaperone protein, an intracellular enzyme (e.g., mRNA encoding anenzyme associated with urea cycle or lysosomal storage disorders)), aprotein associated with the actin cytoskeleton, a protein associatedwith the plasma membrane, a perinuclear protein, a nuclear protein(e.g., a transcription factor), and any other protein involved incellular metabolism, DNA repair, transcription and/or translation). Insome embodiments, cationic lipids having a structure of formula I-c1-aor formula I-g described herein (e.g., cKK-E12) can be used to deliveran mRNA that encodes a transmembrane protein, such as, an ion channelprotein. In some embodiments, cationic lipids having a structure offormula I-c1-a or formula I-g described herein (e.g., cKK-E12) can beused to deliver an mRNA that encodes an extracellular protein including,but not limited to, a protein associated with the extracellular matrix,a secreted protein (e.g., hormones and/or neurotransmitters).

In some embodiments, one or more cationic lipids suitable for thepresent invention may beN-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride or“DOTMA”. (Feigner et al. (Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S.Pat. No. 4,897,355). DOTMA can be formulated alone or can be combinedwith the neutral lipid, dioleoylphosphatidyl-ethanolamine or “DOPE” orother cationic or non-cationic lipids into a liposomal transfer vehicleor a lipid nanoparticle, and such liposomes can be used to enhance thedelivery of nucleic acids into target cells. Other suitable cationiclipids include, for example, 5-carboxyspermylglycinedioctadecylamide or“DOGS,”2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminiumor “DOSPA” (Behr et al. Proc. Nat.'l Acad. Sci. 86, 6982 (1989); U.S.Pat. Nos. 5,171,678; 5,334,761), 1,2-Dioleoyl-3-Dimethylammonium-Propaneor “DODAP”, 1,2-Dioleoyl-3-Trimethylammonium-Propane or “DOTAP”.

Additional exemplary cationic lipids also include1,2-distearyloxy-N,N-dimethyl-3-aminopropane or “DSDMA”,1,2-dioleyloxy-N,N-dimethyl-3-aminopropane or “DODMA”,1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or “DLinDMA”,1,2-dilinolenyloxy-,N -dimethyl-3-aminopropane or “DLenDMA”,N-dioleyl-N,N-dimethylammonium chloride or “DODAC”,N,N-distearyl-N,N-dimethylarnrnonium bromide or “DDAB”,N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide or “DMRIE”,3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propaneor “CLinDMA”,2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethyl-1-(cis,cis-9′,1-2′-octadecadienoxy)propaneor “CpLinDMA”, N,N-dimethyl-3,4-dioleyloxybenzylamine or “DMOBA”,1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane or “DOcarbDAP”,2,3-Dilinoleoyloxy-N,N-dimethylpropylamine or “DLinDAP”,1,2-N,N′-Dilinoleylcarbamyl-3-dimethylaminopropane or “DLincarbDAP”,1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane or “DLinCDAP”,2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane or “DLin-1-DMA”,2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane or“DLin-K-XTC2-DMA”, and2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine(DLin-KC2-DMA)) (see, WO 2010/042877; Semple et al., Nature Biotech. 28:172-176 (2010)), or mixtures thereof. (Heyes, J., et al., J ControlledRelease 107: 276-287 (2005); Morrissey, D V., et al., Nat. Biotechnol.23(8): 1003-1007 (2005); PCT Publication WO2005/121348A1). In someembodiments, one or more of the cationic lipids comprise at least one ofan imidazole, dialkylamino, or guanidinium moiety.

In some embodiments, the one or more cationic lipids may be chosen fromXTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane), MC3(((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate), ALNY-100((3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine)),NC98-5(4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13-tetraazahexadecane-1,16-diamide),DODAP (1,2-dioleyl-3-dimethylammonium propane), HGT4003 (WO 2012/170889,the teachings of which are incorporated herein by reference in theirentirety), ICE (WO 2011/068810, the teachings of which are incorporatedherein by reference in their entirety), HGT5000 (U.S. Provisional PatentApplication No. 61/617,468, the teachings of which are incorporatedherein by reference in their entirety) or HGT5001 (cis or trans)(Provisional Patent Application No. 61/617,468), aminoalcohol lipidoidssuch as those disclosed in WO2010/053572, DOTAP(1,2-dioleyl-3-trimethylammonium propane), DOTMA(1,2-di-O-octadecenyl-3-trimethylammonium propane), DLinDMA (Heyes, J.;Palmer, L.; Bremner, K.; MacLachlan, I. “Cationic lipid saturationinfluences intracellular delivery of encapsulated nucleic acids” J.Contr. Rel. 2005, 107, 276-287), DLin-KC2-DMA (Semple, S. C. et al.“Rational Design of Cationic Lipids for siRNA Delivery” Nature Biotech.2010, 28, 172-176), C12-200 (Love, K. T. et al. “Lipid-like materialsfor low-dose in vivo gene silencing” PNAS 2010, 107, 1864-1869).

In some embodiments, the percentage of cationic lipid in a liposome maybe greater than 10%, greater than 20%, greater than 30%, greater than40%, greater than 50%, greater than 60%, or greater than 70%. In someembodiments, cationic lipid(s) constitute(s) about 30-50% (e.g., about30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) ofthe liposome by weight. In some embodiments, the cationic lipid (e.g.,cKK-E12) constitutes about 30%, about 35%, about 40%, about 45%, orabout 50% of the liposome by molar ratio.

Non-Cationic/Helper Lipids

In some embodiments, provided liposomes contain one or more non-cationic(“helper”) lipids. As used herein, the phrase “non-cationic lipid”refers to any neutral, zwitterionic or anionic lipid. As used herein,the phrase “anionic lipid” refers to any of a number of lipid speciesthat carry a net negative charge at a selected H, such as physiologicalpH. Non-cationic lipids include, but are not limited to,distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), or a mixturethereof.

In some embodiments, such non-cationic lipids may be used alone, but arepreferably used in combination with other excipients, for example,cationic lipids. In some embodiments, the non-cationic lipid maycomprise a molar ratio of about 5% to about 90%, or about 10% to about70% of the total lipid present in a liposome. In some embodiments, anon-cationic lipid is a neutral lipid, i.e., a lipid that does not carrya net charge in the conditions under which the composition is formulatedand/or administered. In some embodiments, the percentage of non-cationiclipid in a liposome may be greater than 5%, greater than 10%, greaterthan 20%, greater than 30%, or greater than 40%.

Cholesterol-Based Lipids

In some embodiments, provided liposomes comprise one or morecholesterol-based lipids. For example, suitable cholesterol-basedcationic lipids include, for example, DC-Choi(N,N-dimethyl-N-ethylcarboxamidocholesterol),1,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem. Biophys.Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997);U.S. Pat. No. 5,744,335), or ICE. In some embodiments, thecholesterol-based lipid may comprise a molar ration of about 2% to about30%, or about 5% to about 20% of the total lipid present in a liposome.In some embodiments, The percentage of cholesterol-based lipid in thelipid nanoparticle may be greater than 5, %, 10%, greater than 20%,greater than 30%, or greater than 40%.

PEGylated Lipids

In some embodiments, provided liposomes comprise one or more PEGylatedlipids. For example, the use of polyethylene glycol (PEG)-modifiedphospholipids and derivatized lipids such as derivatized ceramides(PEG-CER), including N-Octanoyl-Sphingosine-1-[9 Succinyl(MethoxyPolyethylene Glycol)-2000] (C8 PEG-2000 ceramide) is also contemplatedby the present invention in combination with one or more of the cationicand, in some embodiments, other lipids together which comprise theliposome. Contemplated PEG-modified lipids include, but are not limitedto, a polyethylene glycol chain of up to 5 kDa in length covalentlyattached to a lipid with alkyl chain(s) of C₆-C₂₀ length. In someembodiments, a PEG-modified or PEGylated lipid is PEGylated cholesterolor PEG-2K. The addition of such components may prevent complexaggregation and may also provide a means for increasing circulationlifetime and increasing the delivery of the lipid-nucleic acidcomposition to the target cell, (Klibanov et al. (1990) FEBS Letters,268 (1): 235-237), or they may be selected to rapidly exchange out ofthe formulation in vivo (see U.S. Pat. No. 5,885,613).

In some embodiments, particularly useful exchangeable lipids arePEG-ceramides having shorter acyl chains (e.g., C₁₄ or C₁₈). ThePEG-modified phospholipid and derivatized lipids of the presentinvention may comprise a molar ratio from about 0% to about 15%, about0.5% to about 15%, about 1% to about 15%, about 4% to about 10%, orabout 2% of the total lipid present in the liposome.

Polymers

In some embodiments, a suitable delivery vehicle is formulated using apolymer as a carrier, alone or in combination with other carriersincluding various lipids described herein. Thus, in some embodiments,liposomal delivery vehicles, as used herein, also encompass polymercontaining nanoparticles. Suitable polymers may include, for example,polyacrylates, polyalkycyanoacrylates, polylactide,polylactide-polyglycolide copolymers, polycaprolactones, dextran,albumin, gelatin, alginate, collagen, chitosan, cyclodextrins,protamine, PEGylated protamine, PLL, PEGylated PLL and polyethylenimine(PEI). When PEI is present, it may be branched PEI of a molecular weightranging from 10 to 40 kDA, e.g., 25 kDa branched PEI (Sigma #408727).

According to various embodiments, the selection of cationic lipids,non-cationic lipids, PEG-modified lipids and/or polymers which comprisethe lipid nanoparticle, as well as the relative molar ratio of suchlipids to each other, is based upon the characteristics of the selectedlipid(s)/polymers, the nature of the intended target cells, thecharacteristics of the mRNA to be delivered. Additional considerationsinclude, for example, the saturation of the alkyl chain, as well as thesize, charge, pH, pKa, fusogenicity and toxicity of the selectedlipid(s). Thus the molar ratios may be adjusted accordingly.

In some embodiments, the cationic lipids, non-cationic lipids,cholesterol, and/or PEG-modified lipids can be combined at variousrelative molar ratios. For example, the ratio of cationic lipid (e.g.,cKK-E12, C12-200, etc.) to non-cationic lipid (e.g., DOPE, etc.) tocholesterol-based lipid (e.g., cholesterol) to PEGylated lipid (e.g.,DMG-PEG2K) may be between about 30-60:25-35:20-30:1-15, respectively. Insome embodiments, the ratio of cationic lipid (e.g., cKK-E12, C12-200,etc.) to non-cationic lipid (e.g., DOPE, etc.) to cholesterol-basedlipid (e.g., cholesterol) to PEGylated lipid (e.g., DMG-PEG2K) isapproximately 40:30:20:10, respectively. In some embodiments, the ratioof cationic lipid (e.g., cKK-E12, C12-200, etc.) to non-cationic lipid(e.g., DOPE, etc.) to cholesterol-based lipid (e.g., cholesterol) toPEGylated lipid (e.g., DMG-PEG2K) is approximately 40:30:25:5,respectively. In some embodiments, the ratio of cationic lipid (e.g.,cKK-E12, C12-200, etc.) to non-cationic lipid (e.g., DOPE, etc.) tocholesterol-based lipid (e.g., cholesterol) to PEGylated lipid (e.g.,DMG-PEG2K) is approximately 40:32:25:3, respectively. In someembodiments, the ratio of cationic lipid (e.g., cKK-E12, C12-200, etc.)to non-cationic lipid (e.g., DOPE, etc.) to cholesterol-based lipid(e.g., cholesterol) to PEGylated lipid (e.g., DMG-PEG2K) isapproximately 50:25:20:5.

Synthesis of mRNA

mRNAs according to the present invention may be synthesized according toany of a variety of known methods. For example, mRNAs according to thepresent invention may be synthesized via in vitro transcription (IVT).Briefly, IVT is typically performed with a linear or circular DNAtemplate containing a promoter, a pool of ribonucleotide triphosphates,a buffer system that may include DTT and magnesium ions, and anappropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAseI, pyrophosphatase, and/or RNAse inhibitor. The exact conditions willvary according to the specific application.

In some embodiments, for the preparation of mRNA according to theinvention, a DNA template is transcribed in vitro. A suitable DNAtemplate typically has a promoter, for example a T3, T7 or SP6 promoter,for in vitro transcription, followed by desired nucleotide sequence fordesired mRNA and a termination signal.

Desired mRNA sequence(s) according to the invention may be determinedand incorporated into a DNA template using standard methods. Forexample, starting from a desired amino acid sequence (e.g., an enzymesequence), a virtual reverse translation is carried out based on thedegenerated genetic code. Optimization algorithms may then be used forselection of suitable codons. Typically, the G/C content can beoptimized to achieve the highest possible G/C content on one hand,taking into the best possible account the frequency of the tRNAsaccording to codon usage on the other hand. The optimized RNA sequencecan be established and displayed, for example, with the aid of anappropriate display device and compared with the original (wild-type)sequence. A secondary structure can also be analyzed to calculatestabilizing and destabilizing properties or, respectively, regions ofthe RNA.

Modified mRNA

In some embodiments, mRNA according to the present invention may besynthesized as unmodified or modified mRNA. Typically, mRNAs aremodified to enhance stability. Modifications of mRNA can include, forexample, modifications of the nucleotides of the RNA. An modified mRNAaccording to the invention can thus include, for example, backbonemodifications, sugar modifications or base modifications. In someembodiments, mRNAs may be synthesized from naturally occurringnucleotides and/or nucleotide analogues (modified nucleotides)including, but not limited to, purines (adenine (A), guanine (G)) orpyrimidines (thymine (T), cytosine (C), uracil (U)), and as modifiednucleotides analogues or derivatives of purines and pyrimidines, such ase.g. 1-methyl-adenine, 2-methyl-adenine,2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine,N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine,4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine,1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine,7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil),dihydro-uracil, 2-thio-uracil, 4-thio-uracil,5-carboxymethylaminomethyl-2-thio-uracil,5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil,5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil,5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester,5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil,5′-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyaceticacid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil,queosine, .beta.-D -mannosyl-queosine, wybutoxosine, andphosphoramidates, phosphorothioates, peptide nucleotides,methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine. Thepreparation of such analogues is known to a person skilled in the arte.g. from the U.S. Pat. Nos.;4,373,071; 4,401,796; 4,415,732; 4,458,066;4,500,707; 4,668,777; 4,973,679; 5,047,524; 5,132,418; 5,153,319;5,262,530 and 5,700,642, the disclosures of which are incorporated byreference in their entirety.

In some embodiments, mRNAs (e.g., PAH-encoding mRNAs) may contain RNAbackbone modifications. Typically, a backbone modification is amodification in which the phosphates of the backbone of the nucleotidescontained in the RNA are modified chemically. Exemplary backbonemodifications typically include, but are not limited to, modificationsfrom the group consisting of methylphosphonates, methylphosphoramidates,phosphoramidates, phosphorothioates (e.g. cytidine5′-O-(1-thiophosphate)), boranophosphates, positively chargedguanidinium groups etc., which means by replacing the phosphodiesterlinkage by other anionic, cationic or neutral groups.

In some embodiments, mRNAs (e.g., PAH-encoding mRNAs) may contain sugarmodifications. A typical sugar modification is a chemical modificationof the sugar of the nucleotides it contains including, but not limitedto, sugar modifications chosen from the group consisting of2′-deoxy-2′-fluoro-oligoribonucleotide (2′-fluoro-2′-deoxycytidine5′-triphosphate, 2′-fluoro-2′-deoxyuridine 5′-triphosphate),2′-deoxy-2′-deamine-oligoribonucleotide (2′-amino-2′-deoxycytidine5′-triphosphate, 2′-amino-2′-deoxyuridine 5′-triphosphate),2′-O-alkyloligoribonucleotide, 2′-deoxy-2′-C-alkyloligoribonucleotide(2′-O-methylcytidine 5′-triphosphate, 2′-methyluridine 5′-triphosphate),2′-C-alkyloligoribonucleotide, and isomers thereof (2′-aracytidine5′-triphosphate, 2′-arauridine 5′-triphosphate), or azidotriphosphates(2′-azido-2′-deoxycytidine 5′-triphosphate, 2′-azido-2′-deoxyuridine5′-triphosphate).

In some embodiments, mRNAs (e.g., PAH-encoding mRNAs) may containmodifications of the bases of the nucleotides (base modifications). Amodified nucleotide which contains a base modification is also called abase-modified nucleotide. Examples of such base-modified nucleotidesinclude, but are not limited to, 2-amino-6-chloropurine riboside5′-triphosphate, 2-aminoadenosine 5′-triphosphate, 2-thiocytidine5′-triphosphate, 2-thiouridine 5′-triphosphate, 4-thiouridine5′-triphosphate, 5-aminoallylcytidine 5′-triphosphate,5-aminoallyluridine 5′-triphosphate, 5-bromocytidine 5′-triphosphate,5-bromouridine 5′-triphosphate, 5-iodocytidine 5′-triphosphate,5-iodouridine 5′-triphosphate, 5-methylcytidine 5′-triphosphate,5-methyluridine 5′-triphosphate, 6-azacytidine 5′-triphosphate,6-azauridine 5′-triphosphate, 6-chloropurine riboside 5′-triphosphate,7-deazaadenosine 5′-triphosphate, 7-deazaguanosine 5′-triphosphate,8-azaadenosine 5′-triphosphate, 8-azidoadenosine 5′-triphosphate,benzimidazole riboside 5′-triphosphate, N1-methyladenosine5′-triphosphate, N1-methylguanosine 5′-triphosphate, N6-methyladenosine5′-triphosphate, O6-methylguanosine 5′-triphosphate, pseudouridine5′-triphosphate, puromycin 5′-triphosphate or xanthosine5′-triphosphate.

Typically, mRNA synthesis includes the addition of a “cap” on theN-terminal (5′) end, and a “tail” on the C-terminal (3′) end. Thepresence of the cap is important in providing resistance to nucleasesfound in most eukaryotic cells. The presence of a “tail” serves toprotect the mRNA from exonuclease degradation.

Thus, in some embodiments, mRNAs (e.g., PAH-encoding mRNAs) include a 5′cap structure. A 5′ cap is typically added as follows: first, an RNAterminal phosphatase removes one of the terminal phosphate groups fromthe 5′ nucleotide, leaving two terminal phosphates; guanosinetriphosphate (GTP) is then added to the terminal phosphates via aguanylyl transferase, producing a 5′5′5 triphosphate linkage; and the7-nitrogen of guanine is then methylated by a methyltransferase.Examples of cap structures include, but are not limited to, m7G(5′)ppp(5′(A,G(5′)ppp(5′)A and G(5′)ppp(5′)G.

In some embodiments, mRNAs (e.g., PAH-encoding mRNAs) include a 3′poly(A) tail structure. A poly-A tail on the 3′ terminus of mRNAtypically includes about 10 to 300 adenosine nucleotides (SEQ ID NO:9)(e.g., about 10 to 200 adenosine nucleotides, about 10 to 150 adenosinenucleotides, about 10 to 100 adenosine nucleotides, about 20 to 70adenosine nucleotides, or about 20 to 60 adenosine nucleotides). In someembodiments, mRNAs include a 3′ poly(C) tail structure. A suitablepoly-C tail on the 3′ terminus of mRNA typically include about 10 to 200cytosine nucleotides (SEQ ID NO:10) (e.g., about 10 to 150 cytosinenucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10to 40 cytosine nucleotides). The poly-C tail may be added to the poly-Atail or may substitute the poly-A tail.

In some embodiments, mRNAs include a 5′ and/or 3′ untranslated region.In some embodiments, a 5′ untranslated region includes one or moreelements that affect an mRNA's stability or translation, for example, aniron responsive element. In some embodiments, a 5′ untranslated regionmay be between about 50 and 500 nucleotides in length.

In some embodiments, a 3′ untranslated region includes one or more of apolyadenylation signal, a binding site for proteins that affect anmRNA's stability of location in a cell, or one or more binding sites formiRNAs. In some embodiments, a 3′ untranslated region may be between 50and 500 nucleotides in length or longer.

Cap Structure

In some embodiments, mRNAs include a 5′ cap structure. A 5′ cap istypically added as follows: first, an RNA terminal phosphatase removesone of the terminal phosphate groups from the 5′ nucleotide, leaving twoterminal phosphates; guanosine triphosphate (GTP) is then added to theterminal phosphates via a guanylyl transferase, producing a 5′5′5triphosphate linkage; and the 7-nitrogen of guanine is then methylatedby a methyltransferase. Examples of cap structures include, but are notlimited to, m7G(5′)ppp (5′(A,G(5′)ppp(5′)A and G(5′)ppp(5′)G.

Naturally occurring cap structures comprise a 7-methyl guanosine that islinked via a triphosphate bridge to the 5′-end of the first transcribednucleotide, resulting in a dinucleotide cap of m⁷G(5′)ppp(5′)N, where Nis any nucleoside. In vivo, the cap is added enzymatically. The cap isadded in the nucleus and is catalyzed by the enzyme guanylyltransferase. The addition of the cap to the 5′ terminal end of RNAoccurs immediately after initiation of transcription. The terminalnucleoside is typically a guanosine, and is in the reverse orientationto all the other nucleotides, i.e., G(5′)ppp(5′)GpNpNp.

A common cap for mRNA produced by in vitro transcription ism⁷G(5′)ppp(5′)G, which has been used as the dinucleotide cap intranscription with T7 or SP6 RNA polymerase in vitro to obtain RNAshaving a cap structure in their 5′-termini. The prevailing method forthe in vitro synthesis of capped mRNA employs a pre-formed dinucleotideof the form m⁷G(5′)ppp(5′)G (“m⁷GpppG”) as an initiator oftranscription.

To date, a usual form of a synthetic dinucleotide cap used in in vitrotranslation experiments is the Anti-Reverse Cap Analog (“ARCA”) ormodified ARCA, which is generally a modified cap analog in which the 2′or 3′ OH group is replaced with —OCH₃.

Additional cap analogs include, but are not limited to, a chemicalstructures selected from the group consisting of m⁷GpppG, m⁷GpppA,m⁷GpppC; unmethylated cap analogs (e.g., GpppG); dimethylated cap analog(e.g., m^(2,7)GpppG), trimethylated cap analog (e.g., m^(2,2,7)GpppG),dimethylated symmetrical cap analogs (e.g., m⁷Gpppm⁷G), or anti reversecap analogs (e.g., ARCA; m⁷, ^(2′Ome)GpppG, m^(72′d)GpppG,m^(7,3′Ome)GpppG, m^(7,3′d)GpppG and their tetraphosphate derivatives)(see, e.g., Jemielity, J. et al., “Novel ‘anti-reverse’ cap analogs withsuperior translational properties”, RNA, 9: 1108-1122 (2003)).

In some embodiments, a suitable cap is a 7-methyl guanylate (“m⁷G”)linked via a triphosphate bridge to the 5′-end of the first transcribednucleotide, resulting in m⁷G(5′)ppp(5′)N, where N is any nucleoside. Apreferred embodiment of a m⁷G cap utilized in embodiments of theinvention is m⁷G(5′)ppp(5′)G.

In some embodiments, the cap is a Cap0 structure. Cap0 structures lack a2′-O-methyl residue of the ribose attached to bases 1 and 2. In someembodiments, the cap is a Cap1 structure. Cap1 structures have a2′-O-methyl residue at base 2. In some embodiments, the cap is a Cap2structure. Cap2 structures have a 2′-O-methyl residue attached to bothbases 2 and 3.

A variety of m⁷G cap analogs are known in the art, many of which arecommercially available. These include the m⁷GpppG described above, aswell as the ARCA 3′-OCH₃ and 2′-OCH₃ cap analogs (Jemielity, J. et al.,RNA, 9: 1108-1122 (2003)). Additional cap analogs for use in embodimentsof the invention include N7-benzylated dinucleoside tetraphosphateanalogs (described in Grudzien, E. et al., RNA, 10: 1479-1487 (2004)),phosphorothioate cap analogs (described in Grudzien-Nogalska, E., etal., RNA, 13: 1745-1755 (2007)), and cap analogs (including biotinylatedcap analogs) described in U.S. Pat. Nos. 8,093,367 and 8,304,529,incorporated by reference herein.

Tail Structure

Typically, the presence of a “tail” serves to protect the mRNA fromexonuclease degradation. The poly A tail is thought to stabilize naturalmessengers and synthetic sense RNA. Therefore, in certain embodiments along poly A tail can be added to an mRNA molecule thus rendering the RNAmore stable. Poly A tails can be added using a variety of art-recognizedtechniques. For example, long poly A tails can be added to synthetic orin vitro transcribed RNA using poly A polymerase (Yokoe, et al. NatureBiotechnology. 1996; 14: 1252-1256). A transcription vector can alsoencode long poly A tails. In addition, poly A tails can be added bytranscription directly from PCR products. Poly A may also be ligated tothe 3′ end of a sense RNA with RNA ligase (see, e.g., Molecular CloningA Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis(Cold Spring Harbor Laboratory Press: 1991 edition)).

In some embodiments, mRNAs include a 3′ poly(A) tail structure.Typically, the length of the poly A tail can be at least about 10, 50,100, 200, 300, 400 at least 500 nucleotides (SEQ ID NO:11). In someembodiments, a poly-A tail on the 3′ terminus of mRNA typically includesabout 10 to 300 adenosine nucleotides (SEQ ID NO:9) (e.g., about 10 to200 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about10 to 100 adenosine nucleotides, about 20 to 70 adenosine nucleotides,or about 20 to 60 adenosine nucleotides). In some embodiments, mRNAsinclude a 3′ poly(C) tail structure. A suitable poly-C tail on the 3′terminus of mRNA typically include about 10 to 200 cytosine nucleotides(SEQ ID NO:10) (e.g., about 10 to 150 cytosine nucleotides, about 10 to100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides). Thepoly-C tail may be added to the poly-A tail or may substitute the poly-Atail.

In some embodiments, the length of the poly A or poly C tail is adjustedto control the stability of a modified sense mRNA molecule of theinvention and, thus, the transcription of protein. For example, sincethe length of the poly A tail can influence the half-life of a sensemRNA molecule, the length of the poly A tail can be adjusted to modifythe level of resistance of the mRNA to nucleases and thereby control thetime course of polynucleotide expression and/or polypeptide productionin a target cell.

5′ and 3′ Untranslated Region

In some embodiments, mRNAs include a 5′ and/or 3′ untranslated region.In some embodiments, a 5′ untranslated region includes one or moreelements that affect an mRNA's stability or translation, for example, aniron responsive element. In some embodiments, a 5′ untranslated regionmay be between about 50 and 500 nucleotides in length.

In some embodiments, a 3′ untranslated region includes one or more of apolyadenylation signal, a binding site for proteins that affect anmRNA's stability of location in a cell, or one or more binding sites formiRNAs. In some embodiments, a 3′ untranslated region may be between 50and 500 nucleotides in length or longer.

Exemplary 3′ and/or 5′ UTR sequences can be derived from mRNA moleculeswhich are stable (e.g., globin, actin, GAPDH, tubulin, histone, orcitric acid cycle enzymes) to increase the stability of the sense mRNAmolecule. For example, a 5′ UTR sequence may include a partial sequenceof a CMV immediate-early 1 (IE1) gene, or a fragment thereof to improvethe nuclease resistance and/or improve the half-life of thepolynucleotide. Also contemplated is the inclusion of a sequenceencoding human growth hormone (hGH), or a fragment thereof to the 3′ endor untranslated region of the polynucleotide (e.g., mRNA) to furtherstabilize the polynucleotide. Generally, these modifications improve thestability and/or pharmacokinetic properties (e.g., half-life) of thepolynucleotide relative to their unmodified counterparts, and include,for example modifications made to improve such polynucleotides'resistance to in vivo nuclease digestion.

Formation of Liposomes

The liposomal transfer vehicles for use in the present invention can beprepared by various techniques which are presently known in the art. Theliposomes for use in provided compositions can be prepared by varioustechniques which are presently known in the art. For example,multilamellar vesicles (MLV) may be prepared according to conventionaltechniques, such as by depositing a selected lipid on the inside wall ofa suitable container or vessel by dissolving the lipid in an appropriatesolvent, and then evaporating the solvent to leave a thin film on theinside of the vessel or by spray drying. An aqueous phase may then addedto the vessel with a vortexing motion which results in the formation ofMLVs. Uni-lamellar vesicles (ULV) can then be formed by homogenization,sonication or extrusion of the multi-lamellar vesicles. In addition,unilamellar vesicles can be formed by detergent removal techniques.

In certain embodiments, provided compositions comprise a liposomewherein the mRNA is associated on both the surface of the liposome andencapsulated within the same liposome. For example, during preparationof the compositions of the present invention, cationic liposomes mayassociate with the mRNA through electrostatic interactions.

In some embodiments, the compositions and methods of the inventioncomprise mRNA encapsulated in a liposome. In some embodiments, the oneor more mRNA species may be encapsulated in the same liposome. In someembodiments, the one or more mRNA species may be encapsulated indifferent liposomes. In some embodiments, the mRNA is encapsulated inone or more liposomes, which differ in their lipid composition, molarratio of lipid components, size, charge (Zeta potential), targetingligands and/or combinations thereof. In some embodiments, the one ormore liposome may have a different composition of cationic lipids,neutral lipid, PEG-modified lipid and/or combinations thereof. In someembodiments the one or more lipisomes may have a different molar ratioof cationic lipid, neutral lipid, cholesterol and PEG-modified lipidused to create the liposome.

The process of incorporation of a desired mRNA into a liposome is oftenreferred to as “loading”. Exemplary methods are described in Lasic, etal., FEBS Lett., 312: 255-258, 1992, which is incorporated herein byreference. The liposome-incorporated nucleic acids may be completely orpartially located in the interior space of the liposome, within thebilayer membrane of the liposome, or associated with the exteriorsurface of the liposome membrane. The incorporation of a nucleic acidinto liposomes is also referred to herein as “encapsulation” wherein thenucleic acid is entirely contained within the interior space of theliposome. The purpose of incorporating a mRNA into a transfer vehicle,such as a liposome, is often to protect the nucleic acid from anenvironment which may contain enzymes or chemicals that degrade nucleicacids and/or systems or receptors that cause the rapid excretion of thenucleic acids. Accordingly, in some embodiments, a suitable deliveryvehicle is capable of enhancing the stability of the mRNA containedtherein and/or facilitate the delivery of mRNA to the target cell ortissue.

Liposome Size

Suitable liposomes in accordance with the present invention may be madein various sizes. In some embodiments, provided liposomes may be madesmaller than previously known mRNA encapsulating liposomes. In someembodiments, decreased size of liposomes is associated with moreefficient delivery of mRNA. Selection of an appropriate liposome sizemay take into consideration the site of the target cell or tissue and tosome extent the application for which the liposome is being made.

In some embodiments, an appropriate size of liposome is selected tofacilitate systemic distribution of PKU protein encoded by the mRNA. Insome embodiments, it may be desirable to limit transfection of the mRNAto certain cells or tissues. For example, to target hepatocytes aliposome may be sized such that its dimensions are smaller than thefenestrations of the endothelial layer lining hepatic sinusoids in theliver; in such cases the liposome could readily penetrate suchendothelial fenestrations to reach the target hepatocytes.

Alternatively or additionally, a liposome may be sized such that thedimensions of the liposome are of a sufficient diameter to limit orexpressly avoid distribution into certain cells or tissues. For example,a liposome may be sized such that its dimensions are larger than thefenestrations of the endothelial layer lining hepatic sinusoids tothereby limit distribution of the liposomes to hepatocytes.

In some embodiments, the size of a liposome is determined by the lengthof the largest diameter of the lipososme particle. In some embodiments,a suitable liposome has a size no greater than about 250 nm (e.g., nogreater than about 225 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75nm, or 50 nm). In some embodiments, a suitable liposome has a sizeranging from about 10-250 nm (e.g., ranging from about 10-225 nm, 10-200nm, 10-175 nm, 10-150 nm, 10-125 nm, 10-100 nm, 10-75 nm, or 10-50 nm).In some embodiments, a suitable liposome has a size ranging from about100-250 nm (e.g., ranging from about 100-225 nm, 100-200 nm, 100-175 nm,100-150 nm). In some embodiments, a suitable liposome has a size rangingfrom about 10-100 nm (e.g., ranging from about 10-90 nm, 10-80 nm, 10-70nm, 10-60 nm, or 10-5 nm).

A variety of alternative methods known in the art are available forsizing of a population of liposomes. One such sizing method is describedin U.S. Pat. No. 4,737,323, incorporated herein by reference. Sonicatinga liposome suspension either by bath or probe sonication produces aprogressive size reduction down to small ULV less than about 0.05microns in diameter. Homogenization is another method that relies onshearing energy to fragment large liposomes into smaller ones. In atypical homogenization procedure, MLV are recirculated through astandard emulsion homogenizer until selected liposome sizes, typicallybetween about 0.1 and 0.5 microns, are observed. The size of theliposomes may be determined by quasi-electric light scattering (QELS) asdescribed in Bloomfield, Ann. Rev. Biophys. Bioeng., 10:421-150 (1981),incorporated herein by reference. Average liposome diameter may bereduced by sonication of formed liposomes. Intermittent sonicationcycles may be alternated with QELS assessment to guide efficientliposome synthesis.

Pharmaceutical Compositions

To facilitate expression of mRNA in vivo, delivery vehicles such asliposomes can be formulated in combination with one or more additionalnucleic acids, carriers, targeting ligands or stabilizing reagents, orin pharmacological compositions where it is mixed with suitableexcipients. Techniques for formulation and administration of drugs maybe found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co.,Easton, Pa., latest edition.

Provided liposomally-encapsulated or associated mRNAs, and compositionscontaining the same, may be administered and dosed in accordance withcurrent medical practice, taking into account the clinical condition ofthe subject, the site and method of administration, the scheduling ofadministration, the subject's age, sex, body weight and other factorsrelevant to clinicians of ordinary skill in the art. The “effectiveamount” for the purposes herein may be determined by such relevantconsiderations as are known to those of ordinary skill in experimentalclinical research, pharmacological, clinical and medical arts. In someembodiments, the amount administered is effective to achieve at leastsome stabilization, improvement or elimination of symptoms and otherindicators as are selected as appropriate measures of disease progress,regression or improvement by those of skill in the art. For example, asuitable amount and dosing regimen is one that causes at least transientprotein (e.g., enzyme) production.

Suitable routes of administration include, for example, oral, rectal,vaginal, transmucosal, pulmonary including intratracheal or inhaled, orintestinal administration; parenteral delivery, including intradermal,transdermal (topical), intramuscular, subcutaneous, intramedullaryinjections, as well as intrathecal, direct intraventricular,intravenous, intraperitoneal, or intranasal.

Alternately or additionally, liposomally encapsulated mRNAs andcompositions of the invention may be administered in a local rather thansystemic manner, for example, via injection of the pharmaceuticalcomposition directly into a targeted tissue, preferably in a sustainedrelease formulation. Local delivery can be affected in various ways,depending on the tissue to be targeted. For example, aerosols containingcompositions of the present invention can be inhaled (for nasal,tracheal, or bronchial delivery); compositions of the present inventioncan be injected into the site of injury, disease manifestation, or pain,for example; compositions can be provided in lozenges for oral,tracheal, or esophageal application; can be supplied in liquid, tabletor capsule form for administration to the stomach or intestines, can besupplied in suppository form for rectal or vaginal application; or caneven be delivered to the eye by use of creams, drops, or even injection.Formulations containing provided compositions complexed with therapeuticmolecules or ligands can even be surgically administered, for example inassociation with a polymer or other structure or substance that canallow the compositions to diffuse from the site of implantation tosurrounding cells. Alternatively, they can be applied surgically withoutthe use of polymers or supports.

Provided methods of the present invention contemplate single as well asmultiple administrations of a therapeutically effective amount of thetherapeutic agents (e.g., mRNA encoding a PAH protein) described herein.Therapeutic agents can be administered at regular intervals, dependingon the nature, severity and extent of the subject's condition (e.g.,PKU). In some embodiments, a therapeutically effective amount of thetherapeutic agents (e.g., mRNA encoding a PAH protein) of the presentinvention may be administered intrathecally periodically at regularintervals (e.g., once every year, once every six months, once every fivemonths, once every three months, bimonthly (e.g., once every twomonths), monthly (e.g., once every month), biweekly (e.g., once everytwo weeks, every other week), weekly, daily or continuously)

In some embodiments, provided liposomes and/or compositions areformulated such that they are suitable for extended-release of the mRNAcontained therein. Such extended-release compositions may beconveniently administered to a subject at extended dosing intervals. Forexample, in one embodiment, the compositions of the present inventionare administered to a subject twice day, daily or every other day. In apreferred embodiment, the compositions of the present invention areadministered to a subject twice a week, once a week, every 7 days, every10 days, every 14 days, every 28 days, every 30 days, every two weeks(e.g., every other week), every three weeks, or more preferably everyfour weeks, once a month, every six weeks, every eight weeks, everyother month, every three months, every four months, every six months,every eight months, every nine months or annually. Also contemplated arecompositions and liposomes which are formulated for depot administration(e.g., intramuscularly, subcutaneously) to either deliver or release amRNA over extended periods of time. Preferably, the extended-releasemeans employed are combined with modifications made to the mRNA toenhance stability

As used herein, the term “therapeutically effective amount” is largelydetermined based on the total amount of the therapeutic agent containedin the pharmaceutical compositions of the present invention. Generally,a therapeutically effective amount is sufficient to achieve a meaningfulbenefit to the subject (e.g., treating, modulating, curing, preventingand/or ameliorating PKU). For example, a therapeutically effectiveamount may be an amount sufficient to achieve a desired therapeuticand/or prophylactic effect. Generally, the amount of a therapeutic agent(e.g., mRNA encoding a PAH protein) administered to a subject in needthereof will depend upon the characteristics of the subject. Suchcharacteristics include the condition, disease severity, general health,age, sex and body weight of the subject. One of ordinary skill in theart will be readily able to determine appropriate dosages depending onthese and other related factors. In addition, both objective andsubjective assays may optionally be employed to identify optimal dosageranges.

A therapeutically effective amount is commonly administered in a dosingregimen that may comprise multiple unit doses. For any particulartherapeutic protein, a therapeutically effective amount (and/or anappropriate unit dose within an effective dosing regimen) may vary, forexample, depending on route of administration, on combination with otherpharmaceutical agents. Also, the specific therapeutically effectiveamount (and/or unit dose) for any particular patient may depend upon avariety of factors including the disorder being treated and the severityof the disorder; the activity of the specific pharmaceutical agentemployed; the specific composition employed; the age, body weight,general health, sex and diet of the patient; the time of administration,route of administration, and/or rate of excretion or metabolism of thespecific protein employed; the duration of the treatment; and likefactors as is well known in the medical arts.

According to the present invention, a therapeutically effective dose ofthe provided composition, when administered regularly, results inincreased expression of hepatic PAH protein as compared to baselinelevels before treatment. In some embodiments, administering the providedcomposition results in the expression of a PAH protein level at or aboveabout 100 ng/mg, about 200 ng/mg, about 300 ng/mg, about 400 ng/mg,about 500 ng/mg, about 600 ng/mg, about 700 ng/mg, about 800 ng/mg,about 900 ng/mg, about 1000 ng/mg, about 1200 ng/mg or about 1400 ng/mgof total protein in the liver.

In some embodiments, administering provided compositions results inincreased serum PAH protein levels. In some embodiments, administeringprovided compositions results in increased serum PAH protein levels byat least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% ascompared to baseline PAH protein level before treatment. Typically,baseline PAH protein level in serum is measured immediately beforetreatment.

In some embodiments, administering the provided composition results inreduced phenylalanine levels in a biological sample. Suitable biologicalsamples include, for example, whole blood, plasma, serum, urine orcerebral spinal fluid. In some embodiments, administering the providedcomposition results in reduction of phenylalanine levels in a biologicalsample (e.g., a serum, plasma or urine sample) by at least about 5%, atleast about 10%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, or at leastabout 95% as compared to baseline level before treatment. Typically,baseline phenylalanine level is measured immediately before treatment.

In some embodiments, a therapeutically effective dose of the providedcomposition, when administered regularly, results in a reducedphenylalanine level in serum or plasma as compared to the baselinephenylalanine level immediately before treatment. In some embodiments, atherapeutically effective dose of the provided composition, whenadministered regularly, results in a reduced phenylalanine level inserum or plasma as compared to the baseline phenylalanine level insubjects who are not treated. In some embodiments, a therapeuticallyeffective dose of the provided composition, when administered regularly,results in reduction of phenylalanine levels to about 1500 μmol/L orless, about 1000 μmol/L or less, about 900 μmol/L or less, about 800μmol/L or less, about 700 μmol/L or less, about 600 μmol/L or less,about 500 μmol/L or less, about 400 μmol/L or less, about 300 μmol/L orless, about 200 mmol/L or less, about 100 μmol/L or less, or about 50μmol/L in serum or plasma. In a particular embodiment, a therapeuticallyeffective dose, when administered regularly results in reduction ofphenylalanine levels to about 120 μmol/L or less in serum or plasma.

In some embodiments, administering the provided composition results inreduced levels of phenylalanine and or metabolites of phenylalanine(e.g., phenylketone, phenylpyruvate) in the urine.

In some embodiments, one or more neuropsychiatric tests may be used todetermine a therapeutically effective dose. In some embodiments, animprovement on one or more neuropsychiatric tests of at least 10%, 20%,30%, 40% or 50% as compared to either the individual before treatment,or an untreated control individual, indicates that a particular dose isa therapeutically effective amount. In some embodiments, a suitableneuropsychiatric test may be the Inattentive portion of the AttentionDeficit and Hyperactivity Disorder Rating Scale (ADHD-RS) and/or theProfile of Mood States (POMS).

In some embodiments, the therapeutically effective dose ranges fromabout 0.005 to 500 mg/kg body weight, e.g., from about 0.005 to 400mg/kg body weight, from about 0.005 to 300 mg/kg body weight, from about0.005 to 200 mg/kg body weight, from about 0.005 to 100 mg/kg bodyweight, from about 0.005 to 90 mg/kg body weight, from about 0.005 to 80mg/kg body weight, from about 0.005 to 70 mg/kg body weight, from about0.005 to 60 mg/kg body weight, from about 0.005 to 50 mg/kg body weight,from about 0.005 to 40 mg/kg body weight, from about 0.005 to 30 mg/kgbody weight, from about 0.005 to 25 mg/kg body weight, from about 0.005to 20 mg/kg body weight, from about 0.005 to 15 mg/kg body weight, fromabout 0.005 to 10 mg/kg body weight. In some embodiments, the mRNA isadministered at a dose ranging from about 0.1-5.0 mg/kg body weight, forexample about 0.1-4.5, 0.1-4.0, 0.1-3.5, 0.1-3.0, 0.1-2.5, 0.1-2.0,0.1-1.5, 0.1-1.0, 0.1-0.5, 0.1-0.3, 0.3-5.0, 0.3-4.5, 0.3-4.0, 0.3-3.5,0.3-3.0, 0.3-2.5, 0.3-2.0, 0.3-1.5, 0.3-1.0, 0.3-0.5, 0.5-5.0, 0.5-4.5,0.5-4.0, 0.5-3.5, 0.5-3.0, 0.5-2.5, 0.5-2.0, 0.5-1.5, or 0.5-1.0 mg/kgbody weight.

In some embodiments, the therapeutically effective dose is or greaterthan about 0.1 mg/kg body weight, about 0.5 mg/kg body weight, about 1.0mg/kg body weight, about 3 mg/kg body weight, about 5 mg/kg body weight,about 10 mg/kg body weight, about 15 mg/kg body weight, about 20 mg/kgbody weight, about 30 mg/kg body weight, about 40 mg/kg body weight,about 50 mg/kg body weight, about 60 mg/kg body weight, about 70 mg/kgbody weight, about 80 mg/kg body weight, about 90 mg/kg body weight,about 100 mg/kg body weight, about 150 mg/kg body weight, about 200mg/kg body weight, about 250 mg/kg body weight, about 300 mg/kg bodyweight, about 350 mg/kg body weight, about 400 mg/kg body weight, about450 mg/kg body weight, or about 500 mg/kg body weight. In someembodiments, the therapeutically effective dose is administered at adose of or less than about 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, 1.0,0.8, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 mg/kg body weight.

Also contemplated herein are lyophilized pharmaceutical compositionscomprising one or more of the liposomes disclosed herein and relatedmethods for the use of such compositions as disclosed for example, inU.S. Provisional Application No. 61/494,882, filed Jun. 8, 2011, theteachings of which are incorporated herein by reference in theirentirety. For example, lyophilized pharmaceutical compositions accordingto the invention may be reconstituted prior to administration or can bereconstituted in vivo. For example, a lyophilized pharmaceuticalcomposition can be formulated in an appropriate dosage form (e.g., anintradermal dosage form such as a disk, rod or membrane) andadministered such that the dosage form is rehydrated over time in vivoby the individual's bodily fluids.

Provided liposomes and compositions may be administered to any desiredtissue. In some embodiments, the mRNA delivered by provided liposomes orcompositions is expressed in the tissue in which the liposomes and/orcompositions were administered. In some embodiments, the mRNA deliveredis expressed in a tissue different from the tissue in which theliposomes and/or compositions were administered Exemplary tissues inwhich delivered mRNA may be delivered and/or expressed include, but arenot limited to the liver, kidney, heart, spleen, serum, brain, skeletalmuscle, lymph nodes, skin, and/or cerebrospinal fluid.

According to various embodiments, the timing of expression of deliveredmRNAs can be tuned to suit a particular medical need. In someembodiments, the expression of the PAH protein encoded by delivered mRNAis detectable 1, 2, 3, 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, and/or72 hours in serum or target tissues after a single administration ofprovided liposomes or compositions. In some embodiments, the expressionof the PAH protein encoded by the mRNA is detectable 1 day, 2 days, 3days, 4 days, 5 days, 6 days, and/or 7 days in serum or target tissuesafter a single administration of provided liposomes or compositions. Insome embodiments, the expression of the PAH protein encoded by the mRNAis detectable 1 week, 2 weeks, 3 weeks, and/or 4 weeks in serum ortarget tissues after a single administration of provided liposomes orcompositions. In some embodiments, the expression of the protein encodedby the mRNA is detectable after a month or longer after a singleadministration of provided liposomes or compositions.

EXAMPLES

While certain compounds, compositions and methods of the presentinvention have been described with specificity in accordance withcertain embodiments, the following examples serve only to illustrate thecompounds of the invention and are not intended to limit the same.

Example 1 Exemplary Liposome Formulations for hPAH mRNA Delivery andExpression

This example provides exemplary liposome formulations for effectivedelivery and expression of hPAH mRNA in vivo.

Lipid Materials

The formulations described in the following Examples, unless otherwisespecified, contain a multi-component lipid mixture of varying ratiosemploying one or more cationic lipids, helper lipids (e.g., non-cationiclipids and/or cholesterol lipids) and PEGylated lipids designed toencapsulate phenylalanine hydroxylase (PAH) mRNA. Unless otherwisespecified, the multi-component lipid mixture used in the followingExamples were ethanolic solutions of cKK-E12 (cationic lipid), DOPE(non-cationic lipid), cholesterol and DMG-PEG2K.

Messenger RNA Material

Codon-optimized human phenylalanine hydroxylase (PAH) messenger RNA wassynthesized by in vitro transcription from a plasmid DNA templateencoding the gene, which was followed by the addition of a 5′ capstructure (Cap 1) (Fechter, P.; Brownlee, G. G. “Recognition of mRNA capstructures by viral and cellular proteins” J. Gen. Virology 2005, 86,1239-1249) and a 3′ poly(A) tail of approximately 250 nucleotides inlength (SEQ ID NO:12) as determined by gel electrophoresis. 5′ and 3′untranslated regions present in each mRNA product are represented as Xand Y, respectively, and defined as stated (vide infra).

Codon-Optimized Human Phenylalanine Hydroxylase (PAH) mRNA:

-   X—SEQ ID NO:3—Y-   5′ and 3′ UTR Sequences-   X (5′ UTR Sequence)=

[SEQ ID NO.: 4] GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACGY (3′ UTR Sequence)=

[SEQ ID NO.: 5] GGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUCA AAGCU OR(SEQ ID NO.: 6) CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC AAGCU

For example, the codon-optimized human PAH messenger RNA comprised:

(SEQ ID NO: 7) GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACGAUGAGCACCGCCGUGCUGGAGAACCCCGGCCUGGGCCGCAAGCUGAGCGACUUCGGCCAGGAGACCAGCUACAUCGAGGACAACUGCAACCAGAACGGCGCCAUCAGCCUGAUCUUCAGCCUGAAGGAGGAGGUGGGCGCCCUGGCCAAGGUGCUGCGCCUGUUCGAGGAGAACGACGUGAACCUGACCCACAUCGAGAGCCGCCCCAGCCGCCUGAAGAAGGACGAGUACGAGUUCUUCACCCACCUGGACAAGCGCAGCCUGCCCGCCCUGACCAACAUCAUCAAGAUCCUGCGCCACGACAUCGGCGCCACCGUGCACGAGCUGAGCCGCGACAAGAAGAAGGACACCGUGCCCUGGUUCCCCCGCACCAUCCAGGAGCUGGACCGCUUCGCCAACCAGAUCCUGAGCUACGGCGCCGAGCUGGACGCCGACCACCCCGGCUUCAAGGACCCCGUGUACCGCGCCCGCCGCAAGCAGUUCGCCGACAUCGCCUACAACUACCGCCACGGCCAGCCCAUCCCCCGCGUGGAGUACAUGGAGGAGGAGAAGAAGACCUGGGGCACCGUGUUCAAGACCCUGAAGAGCCUGUACAAGACCCACGCCUGCUACGAGUACAACCACAUCUUCCCCCUGCUGGAGAAGUACUGCGGCUUCCACGAGGACAACAUCCCCCAGCUGGAGGACGUGAGCCAGUUCCUGCAGACCUGCACCGGCUUCCGCCUGCGCCCCGUGGCCGGCCUGCUGAGCAGCCGCGACUUCCUGGGCGGCCUGGCCUUCCGCGUGUUCCACUGCACCCAGUACAUCCGCCACGGCAGCAAGCCCAUGUACACCCCCGAGCCCGACAUCUGCCACGAGCUGCUGGGCCACGUGCCCCUGUUCAGCGACCGCAGCUUCGCCCAGUUCAGCCAGGAGAUCGGCCUGGCCAGCCUGGGCGCCCCCGACGAGUACAUCGAGAAGCUGGCCACCAUCUACUGGUUCACCGUGGAGUUCGGCCUGUGCAAGCAGGGCGACAGCAUCAAGGCCUACGGCGCCGGCCUGCUGAGCAGCUUCGGCGAGCUGCAGUACUGCCUGAGCGAGAAGCCCAAGCUGCUGCCCCUGGAGCUGGAGAAGACCGCCAUCCAGAACUACACCGUGACCGAGUUCCAGCCCCUGUACUACGUGGCCGAGAGCUUCAACGACGCCAAGGAGAAGGUGCGCAACUUCGCCGCCACCAUCCCCCGCCCCUUCAGCGUGCGCUACGACCCCUACACCCAGCGCAUCGAGGUGCUGGACAACACCCAGCAGCUGAAGAUCCUGGCCGACAGCAUCAACAGCGAGAUCGGCAUCCUGUGCAGCGCCCUGCAGAAGAUCAAGUAAGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUCAA AGCU

In another example, the codon-optimized human PAH messenger RNAcomprised:

(SEQ ID NO: 8) GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACGAUGAGCACCGCCGUGCUGGAGAACCCCGGCCUGGGCCGCAAGCUGAGCGACUUCGGCCAGGAGACCAGCUACAUCGAGGACAACUGCAACCAGAACGGCGCCAUCAGCCUGAUCUUCAGCCUGAAGGAGGAGGUGGGCGCCCUGGCCAAGGUGCUGCGCCUGUUCGAGGAGAACGACGUGAACCUGACCCACAUCGAGAGCCGCCCCAGCCGCCUGAAGAAGGACGAGUACGAGUUCUUCACCCACCUGGACAAGCGCAGCCUGCCCGCCCUGACCAACAUCAUCAAGAUCCUGCGCCACGACAUCGGCGCCACCGUGCACGAGCUGAGCCGCGACAAGAAGAAGGACACCGUGCCCUGGUUCCCCCGCACCAUCCAGGAGCUGGACCGCUUCGCCAACCAGAUCCUGAGCUACGGCGCCGAGCUGGACGCCGACCACCCCGGCUUCAAGGACCCCGUGUACCGCGCCCGCCGCAAGCAGUUCGCCGACAUCGCCUACAACUACCGCCACGGCCAGCCCAUCCCCCGCGUGGAGUACAUGGAGGAGGAGAAGAAGACCUGGGGCACCGUGUUCAAGACCCUGAAGAGCCUGUACAAGACCCACGCCUGCUACGAGUACAACCACAUCUUCCCCCUGCUGGAGAAGUACUGCGGCUUCCACGAGGACAACAUCCCCCAGCUGGAGGACGUGAGCCAGUUCCUGCAGACCUGCACCGGCUUCCGCCUGCGCCCCGUGGCCGGCCUGCUGAGCAGCCGCGACUUCCUGGGCGGCCUGGCCUUCCGCGUGUUCCACUGCACCCAGUACAUCCGCCACGGCAGCAAGCCCAUGUACACCCCCGAGCCCGACAUCUGCCACGAGCUGCUGGGCCACGUGCCCCUGUUCAGCGACCGCAGCUUCGCCCAGUUCAGCCAGGAGAUCGGCCUGGCCAGCCUGGGCGCCCCCGACGAGUACAUCGAGAAGCUGGCCACCAUCUACUGGUUCACCGUGGAGUUCGGCCUGUGCAAGCAGGGCGACAGCAUCAAGGCCUACGGCGCCGGCCUGCUGAGCAGCUUCGGCGAGCUGCAGUACUGCCUGAGCGAGAAGCCCAAGCUGCUGCCCCUGGAGCUGGAGAAGACCGCCAUCCAGAACUACACCGUGACCGAGUUCCAGCCCCUGUACUACGUGGCCGAGAGCUUCAACGACGCCAAGGAGAAGGUGCGCAACUUCGCCGCCACCAUCCCCCGCCCCUUCAGCGUGCGCUACGACCCCUACACCCAGCGCAUCGAGGUGCUGGACAACACCCAGCAGCUGAAGAUCCUGGCCGACAGCAUCAACAGCGAGAUCGGCAUCCUGUGCAGCGCCCUGCAGAAGAUCAAGUAACGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUCA AGCU

Synthetic codon-optimized human PAH mRNA was transfected into HEK293Tcells and analyzed 24 hours later. Upon cell lysis and processing, humanPAH was successfully detected via western blot analysis (see FIG. 1).

Formulation Protocol

Aliquots of 50 mg/mL ethanolic solutions of cKK-E12, DOPE, cholesteroland DMG-PEG2K were mixed and diluted with ethanol to 3 mL final volume.Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH4.5) of PAH mRNA was prepared from a 1 mg/mL stock. The lipid solutionwas injected rapidly into the aqueous mRNA solution and shaken to yielda final suspension in 20% ethanol. The resulting nanoparticle suspensionwas filtered, diafiltrated with 1×PBS (pH 7.4), concentrated and storedat 2-8° C. Final concentration=1.28 mg/mL PAH mRNA (encapsulated).Z_(ave)=79 nm; PDI=0.12.

Example 2 Administration of hPAH mRNA-Loaded Liposome Nanoparticles

This example illustrates exemplary methods of administering hPAHmRNA-loaded liposome nanoparticles and methods for analyzing deliveredmRNA and subsequently expressed hPAH protein in various target tissuesin vivo.

All studies were performed using male CD-1 mice or PAH knockout mice ofapproximately 6-8 weeks of age at the beginning of each experiment.Samples were introduced by a single bolus tail-vein injection of anequivalent total dose of 1.0 mg/kg (or otherwise specified) ofencapsulated PAH mRNA. Mice were sacrificed and perfused with saline atthe designated time points.

Isolation of Organ Tissues for Analysis

The liver, spleen, kidney and heart of each mouse was harvested,apportioned into separate parts, and stored in either 10% neutralbuffered formalin or snap-frozen and stored at −80° C. for analysis.

Isolation of Plasma for Analysis

All animals were euthanized by CO₂ asphyxiation at designated timepoints post dose administration (±5%) followed by thoracotomy andterminal cardiac blood collection. Whole blood (maximal obtainablevolume) was collected via cardiac puncture on euthanized animals intoserum separator tubes, allowed to clot at room temperature for at least30 minutes, centrifuged at 22° C.±5° C. at 9300 g for 10 minutes, andthe serum was extracted. For interim blood collections, approximately40-504 of whole blood was collected via facial vein puncture or tailsnip. Samples collected from non-treatment animals were used as baselinephenylalanine levels for comparison to study animals.

Phenylalanine Analysis

Phenylalanine levels were measured using a commercially available kit(BioAssay Systems EPHE-100) and by following the manufacturer'sprotocol.

Enzyme-Linked Immunosorbent Assay (ELISA) Analysis—hPAH ELISA

Standard ELISA procedures were followed employing goat polyclonalanti-hPAH antibody (Novus NBP1-52084) as the capture antibody withrabbit anti-hPAH polyclonal antibody (Sigma (HPA02807) as the secondary(detection) antibody. Horseradish peroxidase (HRP)-conjugated goatanti-rabbit IgG was used for activation of the3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution. The reactionwas quenched using 2N H₂SO₄ after 20 minutes. Detection was monitoredvia absorption (450 nm) on a Molecular Device Flex Station instrument.Untreated mouse liver and human hPAH protein were used as negative andpositive controls, respectively.

Example 3 In Vivo Protein Production and Clinical Efficacy

This example demonstrates that administration of hPAH mRNA results insuccessful protein production and clinical efficacy in vivo.

In order to determine if delivered mRNA was successfully translated intoprotein in vivo, quantification of human PAH protein detected in treatedmouse livers was achieved via ELISA-based methods (FIG. 2). FIG. 3further shows that a clear production of human PAH protein was observedwith no cross reactivity with the mouse homolog as confirmed viauntreated wild type mouse livers. Between 6 and 12 hours afteradministration, approximately 300 ng of hPAH protein was detected per mgof total protein in a sample (see FIG. 3).

To determine clinical efficacy, we evaluate the effect of mRNA deliveryin serum phenylalanine levels in PAH knockout mice, a PKU disease model.Phenylalanine levels in untreated PAH knockout mice were extremelyelevated as compared to wild type mice (˜1450 uM vs ˜50 uM). As shown inFIG. 4, upon treatment of these knockout mice with PAH mRNA,phenylalanine levels were brought down to wild type levels within sixhours of dosing. This data demonstrate that hPAH mRNA therapy is highlyeffective in treating PKU.

Example 4 Detection of hPAH mRNA In Vivo

This example demonstrates that following administration of hPAH mRNA,the PAH mRNA is detectable in the liver of mice for at least 72 hours.

Mice were administered a single dose (1.0 mg/kg) of hPAH mRNA-loadedcKK-E12-based lipid nanoparticles, or saline (i.e., control) asdescribed above in Example 2. Mice were sacrificed 30 minutes, 3 hours,6 hours, 12 hours, 24 hours, 48 hours, 72 hours and 7 days followingadministration of the hPAH mRNA and the livers were collected. In situhybridization of the livers was performed to detect the presence of thehPAH mRNA (FIGS. 5A-5I). The presence of hPAH mRNA was observable for atleast 72 hours post-administration (FIGS. 5A-5G). The hPAH mRNA wasdetectable in sinusoidal cells as well as in hepatocytes. These datademonstrate that hPAH mRNA can be detected in the liver for at least 72hours post-administration.

Example 5 Human PAH Protein Levels and Serum Phenylalanine Levels in PAHKnockout Mice after Dose Response Treatment with hPAH mRNA

This example demonstrates a dose response between the amount of hPAHmRNA administered and the amount of both human PAH protein expressed inthe liver and serum phenylalanine levels.

PAH knockout mice were administered a single dose of 0.25 mg/kg, 0.50mg/kg, 0.75 mg/kg or 1.0 mg/kg of hPAH mRNA-loaded cKK-E12-based lipidnanoparticles or saline (i.e., control) as described above in Example 2.A serum sample was collected from the mice prior to the dose (i.e.,pre-dose) and 6 hours after the dose (i.e., post-dose). Mice weresacrificed 6 hours post-administration and the livers were collected.

Human PAH protein levels in the livers were measured by ELISA. Thesedata demonstrate that at all doses, increased levels of hPAH proteinwere detected relative to the control (FIG. 6). These data alsodemonstrate a dose response between the amount of hPAH mRNA administeredand the amount of PAH protein expressed in the liver. For example, miceadministered 1.0 mg/kg of hPAH mRNA expressed approximately 1000 ng ofPAH/mg of total protein while mice administered 0.25 mg/kg of hPAH mRNAexpressed approximately 200 ng of PAH/mg of total protein.

The serum level of phenylalanine was quantified in the pre- andpost-treatment samples (FIG. 7). These data demonstrate a reduction inserum phenylalanine at all treatment doses relative to the pre-dosecontrol, as well as a dose response. For example, mice administered 1.0mg/kg of hPAH mRNA demonstrated lower levels of phenylalanine (i.e.,less than 500 μM) than those administered 0.25 mg/kg (i.e., less than1500 μM).

Example 6 Human PAH Protein and Serum Phenylalanine Levels in PAHKnockout Mice after Treatment with hPAH mRNA for One Month

This example demonstrates that treatment with hPAH mRNA over one monthresults in increased levels of hPAH protein in the liver and decreasedlevels of serum phenylalanine

PAH knockout mice were administered a single dose of 0.5 mg/kg or 1.0mg/kg of hPAH mRNA-loaded cKK-E12-based lipid nanoparticles once perweek for one month or 1.0 mg/kg of hPAH mRNA-loaded cKK-E12-based lipidnanoparticles once every other week for one month, or saline (i.e.,control) as described above in Example 2. Serum was collected from themice prior to the first dose (i.e., pre-dose) and six hours after eachdose. Mice were sacrificed 6 hours after administration of the finaldose on day 29 and the livers were collected.

Human PAH protein levels in the liver were measured by ELISA. These datademonstrate that at all doses, increased levels of hPAH protein weredetected relative to the control (FIG. 8).

The serum level of phenylalanine was quantified in the pre- andpost-treatment samples (FIG. 9). These data demonstrate a reduction inserum phenylalanine at all treatment doses relative to the pre-dosecontrol sample. These data also demonstrate that the higher dose (i.e.,1.0 mg/kg) resulted in lower levels of serum phenylalanine, even whenthe hPAH mRNA was administered every other week.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above Description, butrather is as set forth in the following claims.

We claim:
 1. A method of treating phenylketonuria (PKU), the methodcomprising administering systemically to a subject in need of treatmenta composition comprising an mRNA encoding a phenylalanine hydroxylase(PAH), wherein the mRNA comprises a coding nucleotide sequenceconsisting of SEQ ID NO:3, wherein the mRNA is encapsulated within aliposome with a size of less than 100 nm, and wherein administration ofthe composition at an effective dose and at an administration intervalresults in increased PAH protein level in the liver and in reducedphenylalanine level in the serum as compared to the baseline of PAH andphenylalanine levels before the treatment.
 2. The method of claim 1,wherein the liposome comprises a cationic lipid, a non-cationic lipid, acholesterol-based lipid and a polyethylene glycol (PEG)-modified lipid.3. The method of claim 2, wherein the cationic lipid is selected fromthe group consisting of1,1′-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol)(C12-200), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (MC3),N,N-dimethyl-2,3-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)propan-1-amine(DLinDMA),2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine(DLinKC2DMA, [XTC2]),3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione(cKK-E12),10,13-dimethyl-17-(6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl3-(1H-imidazol-5-yl)propanoate (ICE),(15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine(HGT5000),(4Z,15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine(HGT5001), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),N,N-distearyl-N,N-dimethylammonium bromide (DDAB),1,2-dimyristyloxyproyl-3-dimethyl-hydroxyethyl ammonium bromide (DMRIE),dioleoyloxy-N-[2-sperminecarboxamido)ethyl]-N,N-dimethyl-1-propaniminiumtrifluoroacetate(DOSPA), dioctadecylamidoglycyl spermine (DOGS),1,2-dioleoyl-3-dimethylammonium-propane (DODAP),N,N-dimethyl-(2,3-dioleyloxy)propylamine (DODMA) andN,N-dimethyl-(2,3-dimyristyloxy)propylamine (DMDMA),1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),(2S)-2-(4-((10,13-dimethyl-17-(6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)butoxy)-N,N-dimethyl-3-((9Z,12Z)-octadeca-9,12-dien-1-yloxy)propan-1-amine(CLinDMA),2-[5′-(cholest-5-en-3[betal]-oxy)-3′-oxapentoxy)-3-dimethyl-1-1(cis,cis-9′,12′-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA),1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP),(9Z,9′Z,12Z,12′Z)-3-(dimethylamino)propane-1,2-diylbis(octadeca-9,12-dienoate) (DLinDAP),1,2-dilinoleylcarbamyl-3-dimethylaminopropane (DLinCDAP),2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),2-((2,3-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)propyl)disulfanyl)-N,N-dimethylethanamine(HGT4003), and combinations thereof.
 4. The method of claim 3, whereinthe cationic lipid comprises cKK-E12, which has the structure:


5. The method of claim 2, wherein the cholesterol-based lipid ischolesterol or PEGylated cholesterol.
 6. The method of claim 2, whereinthe cationic lipid constitutes about 30-50% of the liposome by molarratio.
 7. The method of claim 2, wherein the ratio of cationiclipid:non-cationic lipid:cholesterol lipid:PEGylated lipid isapproximately 40:30:25:5 by molar ratio.
 8. The method of claim 1,wherein the liposome comprises a combination selected from the groupconsisting of: cKK-E12, 1,2-dioleyl-sn-glycero-3-phosphoethanolamine(DOPE), cholesterol and 1,2-dimyristoyl-sn-glycerol, methoxypolyethyleneGlycol (DMG-PEG2K); C12-200, DOPE, cholesterol and DMG-PEG2K; HGT4003,DOPE, cholesterol and DMG-PEG2K; or ICE, DOPE, cholesterol andDMG-PEG2K.
 9. The method of claim 1, wherein the mRNA is administered atthe effective dose ranging from about 0.01-5.0 mg/kg body weight. 10.The method of claim 1, wherein the composition is administered once aweek.
 11. The method of claim 1, wherein the mRNA comprises a 5′ UTRsequence of SEQ ID NO:4.
 12. The method of claim 1, wherein the mRNAcomprises a 3′ UTR sequence of SEQ ID NO:5 or SEQ ID NO:6.